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(d) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry-LC3 following transient transfection with GFP-myosin VI. Immunostaining for GFP (green) and cherry (red) was performed and actin was visualized with phalloidin (white). Nuclei were labelled with Hoechst (blue). The arrowheads indicate areas of actin-rich myosin-VI/LC3-positive autophagosomes. Scale bars, 20 μm (a,b,d); 10 μm (c, whole-cell image); 2 μm (c, cropped images).
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Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or H2O2 (500μM) stimulation (E).
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G Co-IP of HEK293T cells transfected to express Hwa-Flag and vector only or plasmids encoding ZNRF3 truncation mutants as indicated in (F). Lysates immunoprecipitated with anti-HA were analyzed by immunoblotting with Flag and HA antibodies. Blots are representative of 3 independent experiments.
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(A B) Neutrophil recovery and morphologic differentiation were assessed in BM (panel A) and PB (panel B) of CCIN mice surviving on day 9. White arrows indicate neutrophil nuclear segmentation.
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C) Full length FLAG-PRKAG2 WT or Δ124 were coexpressed with full length Ulk1 WT and incubated in the presence of 1μM MRT68921. Western blot analysis revealed that Ulk1 overexpression strongly promotes S124 phosphorylation in cells.
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J, K. Liver enzymes (J) alanine aminotransferase (ALT) and (K) alkaline phosphatase (ALP) in plasma at P200 (n=8/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"
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C. In situ hybridization confirms deletion of Slc4a4 in the cortex. GFAP immunostaining intensity was decreased significantly in Slc4a4 icKO at P12 whereas Sox9+ cell number remained unchanged. Scale bar: 100 μm.
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Diversity of Tap-1. Phylogenetic analysis of Tap-1 encoded in 36 V. cholerae strains. Analysis was performed using Raxml (Stamatakis, 2014). The names of Tap-1-harboring strains are indicated in the tree. Three clades are highlighted with different colors. Three representative sequences that cluster to different clades are highlighted with an arrow and used for analysis.
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(c) Tissue lysate from fasted 12-week-old Atg7+/− and control Atg7+/+mice was subjected to immunoblot analysis using anti-LC3 or -p62 antibody. Fold changes of the immunoblot band intensities are shown (middle and right). **P0.01, ***P0.001; Student's t-test, n=3.
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(A) Early stage Drosophila egg chambers showing giant germ-line nurse cells surrounded by E-cadherin-positive follicle cell epithelium. A follicle cell undergoing mitotic rounding is highlighted with an asterisk (*). Scale bar ~3µm. n > 3 independent biological replicates.
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BMDMs of the indicated genotypes were infected with YFP-S. aureus (MOI 10) for 1 h. (H) Representative images of BMDMs stained with an anti-EEA1 antibody (red) and DAPI, 1h post-infection with YFP-S. aureus (green). (I) Quantification of EEA1+ S. aureus-containing phagosomes from panel (G) (n = 27 cells). (J) Representative images of BMDMs stained with an anti-LAMP-1 antibody (red) and DAPI, 1h post-infection with YFP-S. aureus (green). (K) Quantification of LAMP-1+ S. aureus phagosomes from panel (I) (n = 25 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.
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Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. D. Spearman correlation matrix (Benjamini-Hochberg adjusted FDR <5%) between parasitemia levels and immunoregulatory genes differentially expressed between asymptomatic P. falciparum-infected individuals and healthy immune controls. Significant positive correlations are shown in blue and significant negative correlations are shown in red.
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(N) Compared with wild-type animals, levels of ZK1053.4::GFP are dramatically elevated in atg-3 and epg-7 mutants.
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B. Numbers of Venus-positive neurons in the lateral nucleus (LA) and the lateral side of the basal nucleus (BAl) of Naive, Unpaired, and Paired groups. Open and filled bars represent AV-WT and AV-KO mice, respectively. Positive neurons of AV-WT was increased only in the Paired group compared to the Naïve and Unpaired groups in the LA (upper panel). AV-KO was increased in the Unpaired as well as the Paired groups compared to the Naïve group Positive neurons both in the Unpaired and Paired AV-KO mice were increased compared to the same groups of the AV-WT mice In the BAl , positive neurons were increased both in the Unpaired and Paired groups compared to the Naïve group in both the AV-WT and AV-KO mice The error bars represent S.E.M. (n =5/group).
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E. Accumulation of HY5:3HA protein in leaves and roots of reciprocal grafts involving HY5-OE and WT plants grown under white light (200 µmol m-2s-1) for 7 d. Rubisco and β-Actin were used as loading controls for leaves and roots in the western blot analysis, respectively.
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J Frequency of cells with polarized Bem1-GFP signal after incubation at the time and temperature shown. Values are means ± SD, n > 200 cells in each of 3 independent experiments. Paired Student's t-tests were performed.
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F. Bar graph illustrates individual FDG-PET values derived from a whole brain volume of interest. Data represent mean ± SD. 8-15 female mice per group at an average age of 10.7 ± 1.5 months (Grn-/- n = 8, Trem2-/- n = 10, Double-/- n = 10, WT n = 15) were used. Data in F two-tailed student`s t-test was performed.
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Cell death analysis measured by Celigo of PI-positive HT1080 cells, cells were treated with the indicated agents for 24 hrs. (n = 3 biological replicates). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and an unpaired t-test was performed, **** P ≤ 0.0001.
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(B) UASp-GFP-LC3; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP-LC3 pattern. Numerous GFP-LC3 puncta (green) at region 2 within germarium were observed in nutrient-deprived flies. Ovaries were stained with LTR in w1118 flies. Germarium of nutrient-deprived w1118 flies had an increase in punctate LTR staining (red) compared with well-fed germarium.
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A, USP42 alterations in colorectal adenocarcinoma (Cancer Genome Atlas, 2012) (n = 524). Information was retrieved from the cBioPortal in 2017, and updated as displayed in 03/2020.
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H-O ParB‐box mutants are dominant‐negative. Cells of strains DWA362 (ΔminCD, Pspac(hy)‐noc), 363 (ΔminCD, Pspac(hy)‐nocNΔ10), 364 (ΔminCD, Pspac(hy)‐nocQ68R) and 365 (ΔminCD, Pspac(hy)‐nocG86S) were examined after growth for 2 h at 42°C with either no additions (NA) (H-K) or in the presence of 1 mM IPTG (L-O), as indicated. Cell membranes were stained with FM5‐95. Insets show the corresponding phase contrast images. Scale bar, 5 μm.
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Under normal conditions, TCF11/Nrf1 is a short-lived ER-membrane (endoplasmic reticulum) resident protein, which is rapidly subjected to proteasome-mediated degradation following retro-translocation to the cytosol by ER-associated degradation machinery (ERAD). In case of proteasome dysfunction (i.e. proteotoxic stress), the half-life of TCF11/Nrf1 is prolonged and become then a substrate for the NGLY1 and DDI2, thereby giving rise to a C-terminal cleaved fragment that enters into the nucleus. After nuclear translocation, cleaved TCF11/Nrf1 associates with Maf and promotes the expression of proteasomes genes, so that protein homeostasis can be restored. In patients carrying the c.1127+337A>G homozygous PSMC3 variation, defective proteasomes promote a constitutive activation of TCF11/Nrf1 thereby resulting in increased assembly of newly synthetized non-functional proteasomes, which in turn activate TCF11/Nrf1 again. Over-activation of TCF11/Nrf1 results in pathway exhaustion thus rendering patient cells incapable of responding to further proteotoxic stress.
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Example fluorescence traces showing Cas9 transitioning between H and L targets with increasing target distances (right). FRET histograms constructed from events that show fluctuations showing two FRET peaks corresponding to binding to either target. High FRET peak stays the same in each histogram, but lower FRET peak moves to lower FRET values as the distance between targets increases.
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(A) qRT-PCR analysis of PROPEP genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. *, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.
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The heatmap shows the expression of differentially expressed splicing factors in APLNR+ cells, day 5-differentiated cells treated with DMSO or 2.5 nM PLB. The differentially expressed splicing factors were defined as fold change of FPKM > 1.5 between APLNR+ cells and DMSO-treated cells. The heatmap was scaled with Z-Score using the log2(FPKM) expression of differentially expressed splicing factors. n=2 technical replicates.
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(D) Western-blot analysis of Cyclin D1, Annexin A1, and β-actin in RKO cells transfected with control vector or ANXA1. Induction of Cyclin D1 was observed in response to increased Annexin A1. The experiment was repeated twice.
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A. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 and TLR4 in cultured macrophages: RAW264.7 cells cultured in full serum were pre-treated with amlexanox ([50 μM or 100 μM]) (2 hr.), rapamycin [20 ng/mL] (30 min), or Ku-0063794 [1 μM] (30 min) and stimulated -/+poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min). Whole cell lysate (WCL) was immunoblotted as indicated.
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(D) SH-SY5Y cells were similarly transfected as in (A) and processed as in (B). Data from three independent experiments represented as means ± S.E.M., *, p<0.05; one-way ANOVA.
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Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (I) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).
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A-D Proximity between IgM-BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1-min stimulation with the indicated reagents, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.
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F, Microcalorimetric titrations of PXR ligand binding domain (LBD) with FKK5 and FKK6. A single-site binding model was used for fitting the data. The upper panel shows the output signal, dQ/dt, as a function of time. The middle panel shows the integrated heats as a function of the ligand/PXR molar ratio in the cell. The solid line represents the best non-linear least-squares fit of the data. The lower panel shows respective thermodynamic signatures of binding to the PXR ligand binding domain.
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SUM159PT human breast cancer cells were orthotopically injected into the mammary glands of NSG mice. Once SUM159PT tumors reached 250mm3 volume, mice were treated with mock or ASNase (60U per day) for 5 consecutive days (n=3). Following treatment, mice were sacrificed, and blood, mammary glands and tumors were collected and subjected to mass spectrometry to determine the asparagine level. Essential amino acids were used for the raw data normalization. Data are presented as mean ± SD. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.
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Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP. Representative confocal z-stack images showing Gephyrin-positive inhibitory post-synapses in the somas (red) and proximal (green) and distal dendrites (cyan) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the number of Gephyrin-positive inhibitory post-synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice (n = 4 for each group, P*** = 0.0002, P**** < 0.0001).
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(B) IF tissue staining identifying blood vessel invasions (BVI), surrounded by an EC monolayer (arrow) and expanded EC (asterisk), scale bar, 50µm. (C) Quantification of BVI with normal (EC monolayer) and aberrant (expanded EC) phenotype expressed as ratio. N = 5 mice (PBS), n = 3 sections, n = 15 BVI, n = 7 PM (parenchymal metastasis); N = 5 mice (AMD), n = 3 sections, n = 12 BVI, n = 2 PM, ****, p < 0.0001, Fisher's exact test. (
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(A) After 48 hr of CPA injection, mice were treated with low doses of Am80 and/or GCSF for 3 days. Mice were infected with 9 x 106 CFU of S. aureus through intravenous injection on day 4 and sacrificed 16 hr post-infection. Control mice without CPA.
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(C) Co-expression of MBP-Atg19 and prApe1 in Sf21 cells; SDS-PAGE of eluate from amylose beads.
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(E) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Smc5cKO N = 5; two-tailed Mann-Whitney t-test. WT data are from Figure 1H and shown alongside for comparison.
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Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24h.
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A network for the phosphoproteins (encoded genes) and putative kinases family were represented as nodes with color and shapes assigned according to function (e.g., blue squares: metabolic encoded-genes, green triangle: transcription factors or grey hexagon: kinase families). Edges connecting nodes represent functional interactions: transcription factor/TARGET (gray solid arrow), protein-protein (blue solid line), miRNA-RNA (cyan solid line), metabolic (gray solid line with open arrow) or predicted kinase-substrate control (orange dashed line) interactions are coded with indicated colors. Arrow heads in the edges indicate directionality of the interaction. The size of the triangle or hexagon is proportional to the number of targets of the TF or putative kinase family, respectively. The network was grouped by topology using ClusterMaker tool in Cytoscape. The distance between nodes was optimized for visualization purposes.
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(B) Localization in vivo. The indicated strains were grown logarithmically in synthetic complete (SC) medium. After 16 h at 30°C, when the final OD of each culture was about 1, the cells were observed by confocal fluorescence and differential interference (DIC) microscopy. Scale bar: 5 µm.
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(H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with 10 µg/ml cGAMP (H), 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.
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Kaplan-Meier curves for the overall survival of breast cancer patients based on BRCC3 (A), XIST (B), and BRCC3/XIST(C) expression levels. HR: hazard ratio.
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Quantitative expression of Lif mRNA in the colon of DSS-challenged and control mice (n=6 per group).
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Scheme of the ChIRP-MS workflow performed on Xist FL (DOX and noDOX conditions) and Xist ΔB+C (DOX) at day 3 of differentiation; RBP - RNA binding protein.
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MCF10A cells, pre-transfected with the indicated siRNAs, were assayed for migration or invasion in the absence or presence of EGF. Cells that reached the filter's bottom were stained and photographed. Scale bars, 150 μm (migration) and 100 μm (invasion).The results from (A) are depicted as means ± SD (four experiments).
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The cantilever link forms salt-bridges of E74 and E76 with NBD1 residues K89 and R293, respectively.
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B Representative images of N2a cells expressing GFP-Parkin and hTau, hP301L or empty vector (Control) for 24 h, that were then treated with 10 μM CCCP for 4 h. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.
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B The localization of USP26 during meiosis. Immunofluorescence analysis of SYCP3 (red) and USP26 (white) was performed in wild-type spermatocytes. Nuclei were stained with DAPI (blue). The circles indicate the XY body.
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Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, PHD2 and ACTIN protein levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against PHD2.
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Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 126); black bars: BJ cells that were cultured for 7 days in a folate-free medium (n = 131); blue bars: BJ cells cultured for 7 days in a folate-free medium and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 138).Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 360); −folate (n = 372); −folate + AGCT (n = 361).
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D The role of AS-RBM15 in protein translation was assessed by reporter assays. The reporter under control of the CMV promoter contains the 5'UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control. The reporter plasmid was cotransfected with plasmids expressing full-length, exon 1 and Δ5 AS-RBM15 respectively. The vector reporter without 5'UTR is a negative control. (n=3, mean ± SD). P values were calculated by one-way ANOVA test (*p<0.05, **p<0.01 and ***p<0.001).
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(A) qRT-PCR analysis with total RNA isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG-I siRNA or cGAS siRNA and RIG-I siRNA as indicated.
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D,E Markers of the EMT program, (D) ZEB1 (n = 6) are strongly induced in the TGFβ-treated condition.
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(B) Dynamic regulation of fluorescently tagged E-cadherin (E-cad-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of E-cad-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates.
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B Doubling times of diploid yeast strains cultures, grown at 25ºC in YPD medium. N2/Ca and Ca/N2 cells express the same actins but markers used for selection are exchanged. Data are presented as mean +/- SD (n = 3 for all conditions; technical replicates). (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).
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(C) The timing of bipolarity establishment in the acentrosomal pole, chromosome alignment, and anaphase onset Each plot shows the cumulative percentage of each event at individual time points (N=49 cells from two independent experiments).
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786-0 cells were treated with MG132 (10 μM) under aa starvation. Antibodies against GDH1 (C) were used to enrich protein complexes in cells with or without GDH1. IgG was used as a control. WB was performed using the indicated antibodies.
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(A) Oxidation of ketone bodies requires the enzymes SCOT and BDH1 and yields acetyl-CoA which enters the tricarboxylic acid (TCA) cycle.
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A) The position of a CDK1 phosphorylation site, T651CENP-C, is indicated in the diagram of chicken CENPC-CT. EMSA was performed to examine the binding affinities of phosphorylated or non-phosphorylated CENPC-CT (left panel) and CM peptide (right panel) to the CENP-A nucleosome.
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Inducible depletion of USP28 in A-431 upon treatment with doxycycline (1µg/ml) for 96h, western blot (left, VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN (right) was performed.
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E. Confocal microscopy images of MDA-MB-231 cells were co-immunolabeled with antibodies against hTrmt13 and histone H3 (left). The images were captured by GE DeltaVision OMX SR. Co-localization of H3 and hTrmt13 was analyzed by ImageJ (right, n = 20 cells, center line, the median of the data). DAPI staining was included to visualize the cell nucleus (Blue).
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Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated (magenta). In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling. Scale bar: 2 µm.
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(B, C and D) Polyphenols bind to the RhpS-FL protein. The concentration of RhpS-FL protein was consistently measured as 0.6-μM. The binding curves of TA (B), PGG (C) and EGCG (D) are shown. The individual data points were shown (n=2 biological replicates).
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Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress
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E) STED images of representative zygotene-like Stag3 mutant and Smc1β−/− univalents displaying LSAEs. Nuclear spreads were immunostained for SYCP3. Bars, 1μm.
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A Mouse brain tissue was processed for immunohistochemistry by combining anti-lamin B1 (red) and anti-NeuN (green) antibodies. Representative images (maximal Z-projections) showing the distribution of lamin B1 in the striatum of 30-week-old wild-type (WT) and R6/1 mice. Yellow arrowheads show co-localization between lamin B1 and NeuN, and white arrowheads show NeuN negative lamin B1 positive nuclei. Scale bar 50 μm and 25 μm for low and high magnification, respectively.
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f, Quantification of spine density in D1-MSNs, defined as the number of spines normalized to 10µm of dendrite for saline control and after cocaine injection. Mean + SEM; two-way ANOVA, Bonferroni post-hoc test, p* < 0.05; p** < 0.01; p*** < 0.001. n=40 neurons from 4 mice in each experimental group.
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B) Spearman's correlations between baseline performance and rate of decline for all clinical measures.
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f, Scanning electron microscope analysis of primary cilia (marked by arrows) formed in C-19 cells subject to 72 h serum starvation and cycling Atg5+/+ MEFs in normal medium for 24 h. Similar results were observed in three independent experiments.
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C, D. cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax induce neutralising antibody responses that accompany protection. Three weeks after immunisation with 5x104 IU of the indicated HA-VLPs, sera were collected and heat-inactivated. Titres of antibodies capable of neutralising an IAV expressing a matched HA protein were determined by micro-neutralisation (MN) assay (C). The dotted line shows the limit of detection (LOD). Correlation between the MN titers at week 3 and the weight loss at day 4.5 post-challenge is shown in (D).
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C Ubiquitination status of tGnd1‐GFP expressed in S. cerevisiae wt and ubr1Δ san1Δ cells was determined by GFPpull‐down and Western blot analysis using ubiquitin‐specific antibodies. Levels of isolated tGnd1‐GFP after GFPpull‐down were determined by Western blot using GFP‐specific antibodies and are given as a control.
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E) Proliferation assay of CrizR1 cells treated with the indicated concentrations of alvocidib ± 1uM crizotinib for 72h. P=0.2 (n=4).
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J. Representative WB of Skm lipid peroxidation and redox system proteins. Two samples per condition are shown. Each sample contains extracts from 3 mice. Histograms represent quantification (wt, n= 6; LowOXPHOS, n= 6). 4-Hydroxynonenal (4HN), peroxiredoxin 2, 3 and 6 (PRX), superoxide dismutase 1 and 2 (SOD), catalase (CATA) and glutathione reductase (GSR) immunoblots are shown. βF1 is presented as a loading control. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.
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A549 or 16HBE cells were transfected with control or VGLL4 siRNA and treated with MG132 for the indicated times and subjected to immunoblot analysis with the indicated antibodies. Relative IRF2BP2 expression levels were quantified in the right panel
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(C) Detection of endosomal PI(4,5) P2 in cells pre-treated with OSW1 for 1 h in serum-free medium using confocal microscopy and the PLCδ1PH-GFP PI(4,5) P2 reporter. Angiotensin II (AngII, 100 nM) (a Gq and PLC-activating agonist) was used to liberate the PLCδ1PH-GFP PI(4,5) P2 reporter from the PM, which was necessary to detect the endosomal PI(4,5) P2. The signal is transient as the AngII receptors show desensitization. Note the reduced signal in the two PI4K2A K/O clones. Scale bars: 20 µm.
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250 µg of yeast protein extract was processed by R2-P2 using either Fe3+-IMAC, Ti4+-IMAC, Zr4+-IMAC or TiO2 magnetic beads and followed by peptide analysis with DDA-MS. Phosphopeptide enrichment was performed in triplicate. Boxplot depicting distributions of phosphopeptide MS1 signals (n=3). Boxplot depicting distributions of phosphopeptide MS1 signal CVs (n=3).
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(B) Fecal occult blood test in control and cKO mice at indicated timepoints. n=3 at each timepoint.
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K: BM, Trials of NexCre cTKO mice with a direct spatial strategy was strongly reduced compared to LM controls. [strategy F(2,32) = 2,324 ns; geno x strategy F(2,32) = 11.53, p = 0.0002].
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(B) Tangential eye sections through sav3 clones. In sav3 after enclosure many photoreceptors are intact but after 14 days at 29°C most sav mutant photoreceptors have degenerated.
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P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.
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D. In vitro methylation assays were performed as described in (A) with His14-MBP-NSUN3 and in vitro transcribed wildtype mt-tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in (C). Two exposure times of X-ray films are shown: 16 hours (short), 3 days (long).
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B Speed of FM 1-43 penetration in similar experiments. The arrowhead points to one example of ongoing endocytosis during PFA fixation. N = 3-4 independent experiments. The general pattern of FM 1-43 entry was similar to that of propidium iodide. Only the first 10 minutes are shown, to enable an optimal observation of the kinetics of the first stages of FM 1-43 entry. The results parallel those obtained with PI: faster penetration during glyoxal fixation. Scale bar = 40 µm, * p < 0.05, ** p < 0.01
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Protein levels of Sel1L (left panel) and mRNA levels of Fgf21 (right panel) in primary mouse hepatocytes isolated from the tamoxifen-inducible Sel1L-knockout Sel1LERCre mice (2 independent repeats). Hsp90 , loading control for Western blot analysis. Ribosomal L32, loading control for qPCR analysis.
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The absolute number of IL-17A+CD45.1+Vβ11+CD4+ T cells in the spinal cords 11 days after transfer. Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.d)
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a, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 4 h and immunostained for LC3 (LC3B antibody) or ATG12. b, Quantification of results from a (n = 3 per group, **P  0.001). c, Western blot analysis of LC3 (LC3B antibody) from cell lysates from a.
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In a distinct cohort, male GADD45β+/+ (WT; n=5) or GADD45β-/- mice (KO; n= 8) were fed ad libitum (fed) or fasted for 24h (fasted). Serumnon-esterified fatty acids (D) and ketone bodies (E) as well as serum (F) and liver (G) triglycerides (TG) were measured.
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(H) 293T Cells transfected with either empty vector, full length B56δ or indicated mutants were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody.
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(E-G) RAW264.7 cells were transfected with plasmids expressing A20 and RIPK3 (E), or A20 and RIPK3 mutants (G) as indicated, cell lysates were collected for immunoprecipitation and immunoblot. Myc-A20 or Flag-RIPK3 purified from transfected HEK-293T cells was incubated with ATP, E1, E2 and ubiquitin as indicated. The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti-Ub antibody (F). Representative blots from n = 3 biological replicates are shown.
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D. TBC1D31 assembled complex at centrosome positively regulates ciliogenesis and signalling. Ligand (L)-induced stimulation of G-protein coupled receptors (GPCRs) activates the adenylate cyclase (AC) and elevates cAMP levels, which in turn activates PKA. PKA phosphorylates OFD1 at S735, priming the ciliopathy protein to praja2-dependent ubiquitylation and proteasomal degradation. Decreasing the levels of OFD1 negatively impacts on cilium biogenesis and downstream signalling.
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(C) Clearance of α-synuclein inclusions in control (solid bar) or Atg-5 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment are were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01
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Co-IP analysis of impact of MB on interaction between PD-1 and SHP2. 293T cells co-expressing PD-1 and SHP2-FLAG were treated with MB at indicated concentration for 6 hours
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B,D) Cleavage of caspase-3 was detected after 24 h of bortezomib treatment by flow cytometry. The y-axis denotes cell counts and the x-axis represents fluorescence intensity of the APC antibody. The black filled histogram represents untreated control cells, the striated histogram represents 5 nM, and the checkered histogram shows 10 nM bortezomib treated cells.
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A & B) Slices through electron cryotomograms (left panels), central slices through STAs (middle panels) and schematic representations (right panels) of S. oneidensis ΔflgH and P. fluorescens illustrating the presence of the ~55-nm cytoplasmic rings during the assembly of these motors (green arrows). Red dotted circles highlight the complexes in the cryotomograms slices. Scale bars are 50 nm for cryotomogram slices, 20nm for the STA.
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(G) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.
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(c) EGFR degradation in Rubicon knockdown cells. Control or Rubicon shRNA A549 cells were treated with EGF (200 ng ml−1) for the indicated periods and the lysates were subjected to western blotting with the antibodies indicated (left panel). The band intensity was measured in three independent experiments and the mean ± s.d. are shown (right panel).
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Immunolocalization of PS1 in plaques and AVs within dystrophic neurites in AD and PS1/APP mice. Cingulate cortex from 9-mo-old PS1/APP mice immunolabeled with PS1 antibody and NT1 showed that PS1 localized to plaques (A). At higher magnification, anti-PS1 antibodies strongly labeled neuritic profiles that were distributed within the plaque corona (B).
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Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.
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(B) Effects of nutrient starvation on Rubicon, p62 and LC3 in Huh7 cells. Huh7 cells were nutrient-starved for 2 or 6 hours as indicated and lysed for Western-blot analysis. The replicon cells were used as the control for comparison.
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Bar chart showing enrichment of transcription factor binding sites (TFBS) at canonical HIF-1 and HIF-2 sites in HepG2 cells (identified as in Figure 4). TFBSs for 61 additional DNA-binding proteins were determined from ENCODE ChIP-seq data in normoxic HepG2 cells. The significance of the overlap between each set of binding sites and canonical HIF-1 and HIF-2 sites, was determined using a hypergeometric test. Accessible sites determined from ENCODE DNAse-seq data in HepG2 cells was used as a negative control
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(b) Increase in proportion of Ace2+Ctsl+ goblet and club cells with age. Percent of Ace2+Ctsl+ cells (x axis) in different airway epithelial cell types (y axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (SEM).
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