EMBO/sd-character-level-ner huggingface dataset:
Generation of Hops−/− mouse.Figure 1A Schematic diagram of gene targeting of Hops locus. Targeting vector containing lacZ and a ZEN-UB1 cassette was inserted into HOPS gene between exon 2 and 3, flanking with 2 kb 5′ - homology and 2 kb 3′ - homology. B Hops−/− mouse genotyping and protein expression. Genotyping by PCR using genomic DNA showed a 242bp band for wild type (Hops+/+) allele and a 331bp band for mutant (Hops−/−) allele. NTC is Null Template Control (left panel). Genotyping by real time PCR confirmed the absence of Hops mRNA in Hops−/− mice (middle panel). Protein extracts from Hops+/+ and Hops−/− MEFs were analysed by Western Blot using anti-HOPS antibody and anti-α-tubulin antibody as loading control (right panel). Data information: in B data are presented as mean ±SD. C Offspring distribution. Different HOPS genotypes at weaning age (approximately postnatal day 21) from crosses between homozygous knockout mice males and heterozygous females (n=1525 pups). Data information: In C the χ2 test was performed to determine significant differences between the expected and observed ratios of Hops−/− to Hops+/− mice. P < 0.001, by two tailed Student's t-test.
Figure 4(A) AKT phosphorylation in CSCs from MCF-7 cells sorted by CD44. GAPDH protein levels were used as a loading control. (B) Upper panel. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors Wortmannin or LY294002. PTEN, PI3KCA, GSK3 and phospho-AKT Ser-473 protein levels were analyzed by Western blot. GAPDH was used as a loading control. AKT kinase activity was analyzed in these cells by immunoprecipitating phosphorylated AKT proteins with a p-AKT-Ser-473 antibody and using chimeric GSK-3 as a substrate. A two-tailed Student's t test was used to compare the two groups (p<0.05). SEM, standard error of the mean. Lower panel. SKBR3 cells were transiently transfected with a plasmid containing an open reading frame of PTEN, miR-10b or both and phospho-AKT, AKT or tubulin levels analyzed by Western Blot assays. Control is empty vector-transfected cells. (C) Clonogenic ability of miR-10b-depleted CD44+ MCF-7 cells after PI3K pathway inhibition. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors wortmannin or LY294002. Cells transfected with anti-miR-10b or a scrambled control (anti-miR-ctl) were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments. (D) miR-10b overexpression does not induce additional changes in cell colony numbers in response to insulin or PI3K inactivation. MCF-7 stable miR-Scr-OE or miR-10b-OE cells were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.
Figure 1(A) Scheme of identification of Arabidopsis proteins PARylated by PARP2 using proteinmicroarray. Same proteins were spotted as duplicates on the chips. Proteinchips were first incubated with MBP-PARP2proteins in the PARylation reaction buffer with NAD+ (left) or without NAD+ (right). The PARylated proteins indicated as the orange dotted chain were detected with α-PAR antibody hybridization followed by Cy3 fluorophore-conjugated secondary antibody. The fluorescence signals were scanned and imaged by GenePix4100A scanner. (B) Scanned images from a pair of representative protein chips after in vitro PARylation reaction. (C) Subcellular localization of identified candidates based on TAIR prediction. The percentage of each subgroup among the total candidates was presented. (D) GO analysis of identified candidates (dark grey bars) compared to the total proteins on the chip (light grey bars). The proteins were categorized based on the GO annotation by TAIR. X-axis is percentage of genes in a specific functional category; y-axis represents different categories. (E) Confirmation of PARylated proteins with an in vivo PARylation assay. FLAG-tagged candidate proteins were expressed in protoplasts, and immunoprecipitated by α-FLAG antibody after feeding protoplasts with 32P-NAD+. The immunoprecipitated proteins were separated in 10% SDS-PAGE and detected by autoradiography (top panel). The input of FLAG-tagged proteins is shown in an α-FLAGimmunoblot (middle panel), and the protein loading control is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel). The experiments were repeated three times with similar results.
Transition state analysis.Figure 6(A) Schematic representation of the expected Leffler α values if the transition state is structurally similar to the OFS (left) or the IFS (right) or if it assumes an intermediate structure (middle). Color coding is the same in Figure 1. (B-H) LFER analysis. Free energy changes plotted in units of -RT. The activation free energies of the OFS to IFS transitions (blue) and the IFS to OFS transitions (red) were calculated by subtracting the energies of the reference states (black, at the origin) from the energies of the mutated protein variants. The K290A mutation was introduced into the WT, Y204L, A345V/V366A, and Y204/A345V/V366A backgrounds, either substrate-bound or apo (B and G, respectively). R276S/M395R and M362V mutations were introduced into the WT background (C, E, and H). For the transition from Na+/l-Asp-bound to apo state, the free energies measured for the transporters in the presence of Na+ and l-Asp were subtracted from those measured for the apo transporters (D). Mutations at A345, V366 and Y204 sites and their combinations were introduced within the WT and K290A backgrounds (E). LFERs for the perturbations introduced within the WT background use back-calculated WT transition rates (see main text, open symbols). Data are averages over at least three independent repeats and errors are propagated from the standard error of the means in each replicate. (F) Schematic summary of sites where perturbations led to changes in the transition state energy that scaled with the IFS energy (blue) and where they affected only the transition state (grey).
Figure 1A-C Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (see Figure EV1, 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows. D Representative microscopy images showing Fab-PLA results measuring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots and the nuclei are visualized by DAPI staining. Scale bar: 5 μm.E The Fab-PLA results are quantified by Blobfinder software and presented as 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. Data from treated samples were compared to data from resting cells, p-values were calculated by Kruskal-Wallis one-way analysis of variance (ANOVA).
Figure 5Western blot analysis of Methionine Sulfoxidation (MetO), Methionine sulfide reductase B2 (MsrB2) and Parkin in human Healthy Control (HC) and Diabetic Mellitus (DM) platelets. P.C. is a BSA-MetO positive control (Cayman Chemical Co.). Quantitation of western blot analysis shown in Fig 1C. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (MetO; **p=0.0003, MsrB2; *p=0.0186, Parkin; **p=0.0003 vs. HC). Western blot analysis of Parkin and Parkin aggregation in human Healthy Control (HC) (n=3) and Diabetic Mellitus (DM) (n=12) platelets. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (modified parkin *p=0.035 vs. HC). Actin served as the loading control. Parkin IP was performed in HC, HC plus H2O2, and DM patients. MetO sites were analyzed by mass spectrometry using the Mascot program Representative mass spectrometry analysis demonstrating identification of MetO192 peptide on Parkin. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Modulation of β‐catenin expression regulates autophagosome number.Figure 1(A-C) Measurement of autophagy during nutrient starvation in HT29 cells stably expressing YFP-LC3. (A) Increased LC3 puncta per cell following β‐catenin knockdown with siRNA compared with a non‐targeting (NT) control siRNA. Columns show autophagosome numbers (mean±s.e.m., n>200 cells in >20 fields of view per experiment of three independent treatments) assessed under normal and starved (2 h) conditions with β‐catenin or non‐targeting (NT) siRNA. Representative images (B) of YFP-LC3 puncta (green) and DAPI nuclei staining (blue) are shown. (C) Upper panel: western blotting showed increased LC3‐II expression in HT29 cells with β‐catenin knockdown compared to control cells after 2 h starvation. Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.011 in nutrient conditions, *P=0.035 under starvation). (D and E) HCT116 β‐cateninWT/− cells overexpressing a control or β‐cateninS33Y plasmid nutrient starved for 24 h. (D) Immunostaining of LC3 (green) with DAPI nuclei staining (blue) showed decreased LC3 puncta with β‐cateninS33Y overexpression. (E) By western blotting, LC3‐II decreased with β‐cateninS33Y overexpression (upper panel). Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.015). (F) Comparison of isogenic β‐cateninWT/− and β‐cateninHCT116 cells. Upper panel: western blotting showed decreased LC3‐II in HCT116β‐catenin cells. Lower panel: western blotting confirmed by quantification using densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent treatments, *P=0.046).Source data for this figure is available on the online supplementary information page.
Figure 3Schematic showing the generation of a translocation intermediate with SecA dN20::H6 (purple) recruited to SecYEG liposomes containing Ni-modified lipids (cyan circles). Addition of imidazole dissociates SecA dN20::H6, which can then be replaced by WT or mutant SecA (tan). Imidazole (imi) and either WT or mutant SecA were added to translocation intermediates (IM) pre-formed with SecA dN20::H6. The samples were incubated for the indicated times before proteolysis with proteinase K (Prot. K). The gel is representative of two replicates, except for the experiment with the R792G/K797G mutant and the 30-min time points. Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to different SecA mutants as shown in the inset box. As in (B), but ATPγS was added together with imidazole. The gel is representative of two replicates. Quantification of (E) as in (C). As in (D), but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA. The gel is representative of two replicates.   Quantification of (G) as in (F), except that both the intensities of the IM (darker colors) and FL (lighter colors) bands were measured.   Schematic of the residues at the tip of the SecA two-helix finger. The conserved tyrosine Y794 is indicated in blue, and the conserved basic residues R792 and K797 are in red.
Figure 2(A) Western blot showing loss of endophilin A2 protein after SH3GL1 targeting in Ramos cells using 2 independent sgRNAs, compared to 2 non-targeting sgRNAs. (B) Soluble anti-IgM F(ab')2 antigen internalization at 20 min in Ramos cells targeted with indicated sgRNAs. P, significance in one-way ANOVA versus control. Plot shows mean and SEM of 7 independent sgRNA infections. (C) Validation of gene candidates affecting soluble antigen uptake in primary mouse B cells. Follicular B cells isolated from CRISPR-targeted bone marrow chimera mice internalize anti-mouse IgM F(ab')2. Data show mean and SEM of N = 3 mice. P, statistics from 2-way ANOVA; at 5 min all 3 genotypes show p<0.0005 versus control; at 15 min p values are indicated in corresponding color. (D) Antigen internalization from PMS in SH3GL1-targeted Ramos cells. ****p<0.0001 in one-way ANOVA. N = 547, 389 and 136 cells respectively, from 2 experiments. (E) Internalization of soluble Fab or F(ab')2 fragments in Ramos cells targeted with indicated sgRNAs. *p=0.0124 **p=0.0092 ***p=0.0004 using 2-way ANOVA. Data show mean ± SEM, from 6 biological replicates across 2 experiments. (F) TIRF images of Ramos cells transduced with endophilin A2-GFP and mCherry-ClathrinLC on antigen-loaded PMS. Scale bar = 5 μm. White arrowheads highlight endophilin A2 colocalization with antigen clusters. (G) Percent of endophilin A2 and clathrinLC spots colocalizing with each other. Levels of random spot colocalization were calculated using randomized spot coordinates. Data show mean and SEM from 107 cells analyzed from 4 experiments; statistics from one-way ANOVA. (H) Percentage of surrogate antigen spots colocalizing with endophilin A2, clathrin LC or both markers. Levels of random spot colocalization were calculated using randomized spot coordinates. Data show mean and SEM from 107 cells analyzed from 4 experiments; statistics from one-way ANOVA. (I) Colocalization of surrogate antigen clusters with endophilin A2-GFP following deletion of clathrin adaptors EPN1 or PICALM1. Data show mean and SEM from 23-29 cells. (J) IgM-BCR immunoprecipitation to investigate recruitment of endophilin A2 to activated BCRs. Anti-endophilin A2 blots detect overexpressed endophilin A2-GFP in streptavidin immunoprecipitates and total supernatants from anti-IgM-biotin-stimulated Ramos and DG75 cells. Representative experiment and quantification from 3 independent pulldowns in WT, BLNK- or GRB2-knockout (KO) DG75 cells. Pulldown intensity is calculated as a percentage of total endophilin A2 protein and normalized to WT in each experiment. Data show mean and SEM; p values calculated using one-way ANOVA. (K) Recruitment of endophilin A2 to antigen clusters quantified as mean antigen intensity in endophilin A2 clusters relative to mean antigen intensity in the entire synapse. N = 30, 29 and 36 cells respectively, from 2 experiments. P, significance in Kruskal-Wallis test with multiple comparisons.
SvbREP suppresses stem cell tumors in the gut epitheliumA esgts midguts expressing GFP alone (control), or expressing Notch-RNAi, Notch-RNAi + svb-RNAi, and Notch-RNAi + SvbREP.Figure 6esgts midguts expressing GFP alone (control), or expressing Notch-RNAi, Notch-RNAi + svb-RNAi, and Notch-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells. esgts midguts expressing GFP alone (control), or expressing STAT-RNAi, STAT-RNAi + svb-RNAi and STAT-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells. esgts midguts expressing GFP alone (control), or expressing Wg, Wg + OvoB, Wg + SvbREP, and Wg + svb-RNAi. In closeup views, nuclei are in purple.
a) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h.figf3(a) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Calpain activity was detected by immunoblotting with anti-calpain small subunit antibody. Densitometry analysis is relative to actin. Error bars show s.e.m. (b) SK-N-SH cells transfected with EGFP-HDQ74 for 4 h, then pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 48 h post-transfection, were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval. (c) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Endogenous LC3-II levels were detected by immunoblotting with anti-LC3 antibody. (d) Comparison between calpain activity, EGFP-HDQ74 aggregation and autophagosomes as seen in a-c. The control condition for all the assessments was set at 100%. Calpastatin lowered the increased calpain activity and EGFP-HDQ74 aggregation caused by (±)-Bay K8644 in a dose-dependent manner with a simultaneous increase in LC3-II levels (autophagosomes). (e) COS-7 cells transfected with empty vector (pcDNA3.1) or wild-type PLC-ε for 4 h and treated with or without 50 μM calpeptin for 48 h post-transfection were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
Figure 4A. OT-II WT cells were activated with OVA323-339, alone or with TAK-779. After three days, antigen and TAK-779 were removed and lymphoblasts expanded in IL-2-containing medium. TCR nanoclustering was analyzed in anti-CD3ε-labeled surface replicas of day 10 lymphoblasts. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of WT CD4+ T cells alone or with TAK-779. Bottom, quantification of gold particles in clusters of the indicated size. Inset, distribution of gold particles between clusters of one, two, three, four or more than four particles in vehicle- (gray bars; n = 5 cells, 11266 particles) and TAK-779-treated cells (black; n = 6 cells, 5138 particles). B. OT-II WT cells were activated with OVA323-339, and TAK-779 was added at days 3, 5, and 7 after antigen removal. Analysis as above, untreated (gray bars; n = 5 cells, 6400 particles) and TAK-779-treated cells (black; n = 6 cells, 7153 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis. C. OT-II WT naïve cells were activated with antigen in the presence or not of the CXCR4 inhibitor AMD3100. Left, representative EM images showing gold particle distribution in the cell surface replicas. Right, analysis of gold particles in clusters as above, vehicle- (gray bars; n = 6 cells, 12339 particles) and AMD3100-treated cells (black; n = 7 cells, 17059 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.
Sumoylation of FXR blocks its interaction with RXRα and increases its interaction with NF‐κB A Effects of expression of either FXR‐WT or FXR‐K277R on the mRNA levels of the indicated genes in hepatocytes isolated from FXR‐KO mice.Figure 6A Effects of expression of either FXR‐WT or FXR‐K277R on the mRNA levels of the indicated genes in hepatocytes isolated from FXR‐KO mice. Values are presented as mean ± SEM (n = 6); two‐tailed Student's t‐test, **P 0.005, NS, not significant. B Reporter assays: HepG2 cells were transfected with plasmids as indicated and treated with GW4064, and luciferase activities were measured. Values are presented as mean ± SEM (n = 3); two‐tailed Student's t‐test, *P 0.05, **P 0.005, NS, not significant. C Experimental outline for in vitro CoIP studies. D Effects of SUMOylation of FXR on its interaction with RXRα. E FXR modeling analysis: The predicted structure of FXR and RXR bound to DNA is shown. The positions of K217 and K277 in FXR are indicated. K277 in the LBD of FXR is located in the interface with the RXR DBD (left). SUMO2 modification (dimers of SUMO2 are shown) of the LBD of FXR would block the interaction of FXR with RXRα (right). Modeling was done using the RXR/PPARγ/DNA (3DZU) model with PPARγ replaced by FXR (1OSV). F, G Effects of the mutation, K277R, on the interaction of FXR with the p65 subunit of NF‐κB (F) or SMRT (G) detected by CoIP. H Effects of SUMOylation of FXR on its interaction with p65. FXR was SUMOylated in vitro, and the interaction of SUMO‐FXR with p65 was detected by CoIP. I, J A schematic of the domains of p65 or FXR that were fused to GST is shown on the top. The interaction between FXR and p65 or their domains was detected by GST pull‐down followed by IB.
Figure 4A, Immunofluorescence confocal microscopy of ARPE-19 cells overexpressing TFEB-FLAG-WT or TFEB-FLAG-C212A showing the subcellular distribution of recombinant TFEB in response to treatments with EBSS for 4 h or 250 μM NaAsO2 for 1 h. Scale bars, 10 μm. B, Quantification of the nuclear localization of recombinant TFEB in ARPE-19 cells treated with 250 μM NaAsO2 for 1 h or EBSS for 4h and incubated in complete medium (refeed) for the indicated times. Data are presented as mean ± SD using two-way ANOVA, (ns) not significant as compared to the same treatment in TFEB-WT overexpressing cells, with >200 cells counted per trial from three independent experiments. C, Immunoblot analysis of protein lysates from MEF cells treated with EBSS, 250 nM Torin-1 or 100 μM NaAsO2 for 1 h, subjected to subcellular fractionation and run under non-reducing condition. D, Relative quantitative RT-PCR analysis of the mRNA expression of autophagy- (UVRAG), mitochondria- (PGC-1α) and lysosome (MCOLN1, ATPV1C1 and HEXA)-related genes in ARPE-19 cells overexpressing TFEB-FLAG-WT or TFEB-FLAG-C212A. Data are presented as mean ± SD using two-way ANOVA, (ns) not significant as compared to the same gene in TFEB-WT overexpressing cells, from three independent experiments.
Effects of missense mutations and small-molecules on PPI detection with LuTHy.Figure 4A Live-cell bioluminescence imaging (BLI) of HEK293 cells co-producing the indicated fusion proteins. mCitrine was excited at 500 nm, emitted fluorescence was detected at 535 nm. After substrate addition, short (460) and long (535) band-pass (BP) filters in a dual-view adapter were used to detect the emitted luminescence simultaneously at the respective wavelengths. BRET images were calculated by dividing the 535 BP by the 460 BP images using ImageJ. Scale bar = 20 μm. B Quantification of the rapamycin-induced interaction between NL-FKBP12 and PA-mCit-FRB. Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin. After an additional incubation for 4 h BRET was quantified upon substrate addition. After cell lysis in buffer with indicated rapamycin concentrations, LuC ratios were determined. EC50 values were obtained with non-linear curve fitting. Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and After an additional incubation for 4 h BRET was quantified upon substrate addition. LuC ratios were determined. EC50 values were obtained with non-linear curve fitting. C Effects of Nutlin-3 on the interaction between NL-MDM2 and PA-mCit-p53. for 6 h treatment with Nutlin-3. D Time resolved HSF1 oligomerization by Hsp90 inhibition. HEK293 cells co-producing NL-HSF1 and PA-mCit-HSF1 for 24 h were transferred to 384-well plates containing Ganetespib or Geldanamycin to reach a final concentration of 1 µM. Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control. Data are the mean of two biological replicates. All values are mean ± sem. Significance was calculated by two-way ANOVA followed by Dunnett's multiple comparison's post-hoc test. E Schematic depiction of a heat shock experiment. HEK293 cells co-producing NL-HSF1/PA-mCit-HSF1 for 48h were subjected to a heat shock at 42°C for 6 h, after which the cells were recovered at 37°C for 24h. Luminescence was measured at the indicated time points: immediately before the heat shock, during the heat shock at 90, 180 and 360 min and after a recovery phase of 24 h. Calculated cBRET ratios were normalized to a control plate that was not heat treated. All values are mean ± sem from three independent experiments. Significance was calculated by one-way ANOVA followed by Dunnett's multiple comparisons post-hoc test; *p<0.05.