EMBO/sd-character-level-ner huggingface dataset:
LDB2/Ldb2 forms complexes with known transcription regulators A-E.Figure 7Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LDB2 (A) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively. Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LHX2 (B) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively. Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LMO2/3/4 (C) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively. Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates , LDB2 vs. SSBP2/3/4 (D) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively. Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. RLIM (E). LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively. F. Coimmunoprecipitation of endogenous Ssbp3 and Lmo3 with Ldb2. Immunocomplexes from mouse brain lysate using an antibody against Ldb2, Lmo3 or Ssbp3, or a control antibody were analyzed by western blotting using an anti-Ldb2 (left) or anti-Ssbp3 (right) antibody.
/PLK1 phosphorylation-dependent complex formation(A-C) (A) Schematic outline of EBNA2, its dimerization domains (END, DIM), the region used by CBF1 to recruit EBNA2 to DNA (WW), the C-terminal transactivation domain (TAD), the nuclear localization signal (N) and the EBNA2 fragments used to map the PLK1 docking site (UniProt ID: P12978.Figure 2(A) Schematic outline of EBNA2, its dimerization domains (END, DIM), the region used by CBF1 to recruit EBNA2 to DNA (WW), the C-terminal transactivation domain (TAD), the nuclear localization signal (N) and the EBNA2 fragments used to map the PLK1 docking site The panel on the right summarizes the results of the co-immunoprecipitations in (B) Transfection of DG75 B cells and immunoprecipitation of (B) HA-tagged or for smaller fragments (C) GFP-tagged EBNA2 fragments to co-precipitate endogenous PLK1. Total protein lysates and immunoprecipitates (IP) were analyzed by Western blotting (WB). (D) Multiple sequence alignment (left) and superposition (right) of several phosphopeptides present in published crystal structures of PLK1 PBD (phosphorylated residues stained red). Crystal structures of the PBD in complex with peptides show that the positively charged groove of PBD docks in a similar mode to the negatively charged phosphopeptides. Potential residues of EBNA2, which might be a PBD docking site, are listed below. (E) Immunoprecipitation of HA-tagged EBNA2 mutant ST266AV, TSS377VAA and SPSS467APAA using HA-specific antibodies. (F) ITC thermogram of PLK1 PBD titrated with the peptide PNTSSPS or the phosphopeptide PNTSpSPS of EBNA2. (G) GST-pulldown of PLK1 from total cellular extracts using Cyclin B/CDK1 phosphorylated GST-EBNA2 region 342-422. (H) Co-immunoprecipitation of transfected EBNA2 wt or S379A and endogenous PLK1.
a) Mouse fibroblasts knockeddown for LAMP-2A and transfected with KFERQ–PS–CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h.f4(a) Mouse fibroblasts knockeddown for LAMP-2A and transfected with KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Representative images of cells also immunostained for LAMP-1 are shown. (b, c) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were photoconverted and maintained for 16 h in media supplemented (+) or not (−) with serum in the presence of 3-methyladenine (3-MA) to inhibit macroautophagy. (b) Representative images of the 3-MA-treated cells. (c) Quantification of the number of green fluorescence puncta per cell in 3-MA treated or not cells maintained in the presence (white bars) or absence (black bars) of serum. Values are mean+s.e. of 3 different experiments with >50 cells counted per experiment. No significant differences were detectable between samples supplemented or not with 3MA. Scale bars, 5 μm.
Figure 2(A) Main VAV1 mutation functional subclasses according to the data obtained in Figure 1. The color code of each subclass shown at the top will be used in panels B and C to facilitate the identification of each of them. The percentage of each mutant subclass (relative to all the deregulated VAV1 mutants tested) is also indicated at the top. The GOF and LOF effects of each mutant subclass are shown in red and blue color (boxes and arrows). WT-like activity is depicted as green arrows and boxes. The domains targeted by the mutations are shown in gray boxes at the bottom of each subclass. The tumors where the mutations have been found are shown at the bottom of each subclass. L, lung cancer; P, peripheral T cell lymphoma. (B) Distribution of VAV1 mutation functional subclasses in the indicated tumor subtypes. The mutation subclasses are indicated following the color codes shown in the inset on the right. (C) Type of mutations associated with the indicated functional subclasses (which are indicated following the color codes shown in the inset present in panel B).
Figure 3A LC-MS/MS analysis of S1P-binding proteins. Total protein lysate was subjected to coimmunoprecipitation (Co-IP) with normal IgG or S1P antibody. The purified protein complex was separated by SDS-PAGE and then subjected to silver staining. The arrows indicate the bands containing S1P, ETFA and ETFB. The differential bands were then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). B Role of the S1P-ETFA-ETFB interaction network in mitochondrial and nonmitochondrial protein systems. C S1P interacts with endogenous ETFA and ETFB in HaCaT cells. Total protein lysate was subjected to Co-IP with normal IgG, ETFA (left panel) or ETFB (right panel) antibody. The interaction between S1P and ETFA/ETFB was then detected by immunoblotting. D The direct interaction between S1P and ETFA-ETFB was validated by a GST pull-down assay. Left panel: in vitro-translated S1P was pulled down by purified GST-ETFA fusion protein. Right panel: in vitro-translated S1P was pulled down by purified GST-ETFB fusion protein. E Confocal immunofluorescence demonstrated that S1P colocalized with ETFA and ETFB in HaCaT cells. The black arrowheads indicate the colocalization (white) of S1P (green), ETFA (red) and ETFB (purple). Scale bars: 20 μm (panels 1-5); scale bars: 2 μm (panel 6). F Three-dimensional structure of the S1P-ETFA-ETFB-FAD complex in stereo. The structures of S1P (magenta), ETFA (light blue), ETFB (green) and FAD (red) are depicted in carbon.
a, Skeletal muscle.figf3a, Skeletal muscle. Muscle extracts from patients 1-6 and 10 and a control were blotted and labelled with antibodies against LAMP-2 (top panel) and β-actin (bottom panel). LAMP-2 is deficient in all muscles examined except the sample from patient 1 that showed a trace amount of protein, which is probably the LAMP-2a isoform. In support of this idea, LAMP-2b is the principal LAMP-2 species in skeletal muscle6. Numbers denote patients as in Table 1; C, control. b, Heart. Protein was extracted from cardiac muscle of patient 4 and a control, blotted onto a nitrocellulose membrane, and labelled with the antibodies against LAMP-2 (too panel) and β-actin (bottom panel). LAMP-2 is deficient also in cardiac muscle. C, control; 4, patient 4.
PDK inhibition via DCA treatment eliminates the oscillation of glycolytic flux into TCA cycle and inhibits cellular progression through S phase.Figure 6(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in citrate m+2 (a) from isotopic glucose (representing glycolytic flux into TCA cycle). (a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in acetyl-CoA m+2 (b) from isotopic glucose (representing glycolytic flux into TCA cycle). It further inhibits oscillations in citrate m+5/m+4 ratio (c) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux). It further inhibits oscillations in acetyl-CoA m+2 (d) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux). (e) The fraction of cells in G1, S, and G2/M phases in synchronized HeLa cells after 3 hour treatment with DCA (normalized by measurements in untreated control cells). (f) The fraction of cells in G1, S, and G2/M phases in non-synchronized HeLa and HCT-116 cells after 24 hour treatment with DCA (normalized by measurements in untreated control cells). As shown, DCA significantly increases the fraction of cells in S phase, inhibiting cellular progression into G2 phase; showing mean and s.d. of n=3 for all isotopic labeling forms and FACS measurements (a-f). Statistical significance of changes in the fraction of cells in each cell cycle phase following DCA treatment is calculated based on two-tailed, unequal variance t-test (e-f).
Figure 2(A) Scatter plot of gene expression differences between Pax5Jak2/+ and Pax5+/+ pro-B cells, which were isolated from the bone marrow of 3-week-old mice prior to RNA-sequencing. The expression data of individual genes (indicated by dots) are plotted as mean normalized rlog (regularized logarithm) values. Genes with an expression difference of > 2-fold, an adjusted P value of < 0.05 and a TPM value of > 5 (in at least one cell type) are colored in blue and red, corresponding to upregulation or downregulation in Pax5Jak2/+ pro-B cells (Dataset EV1). (B) Bar graph indicating how many of the up- or down-regulated genes in Pax5Jak2/+ pro-B cells correspond to repressed (orange) or activated (green) Pax5 target genes identified in pro-B cells (C) Scatter plot of gene expression differences between Pax5Jak2/+ and Pax5+/- Cdkn2ab+/- B-ALL cells of tumors that were isolated from lymph nodes prior to RNA-sequencing. Genes with an expression difference of > 3-fold, an adjusted P value of < 0.05 and a TPM value of > 5 (in at least one tumor cell type) are colored in blue and red, corresponding to upregulation or downregulation in Pax5Jak2/+ B-ALL cells (D-G) GSEA analysis of 327 repressed (D) or 330 activated (F) Pax5 target genes identified in pro-B cells as compared with the ranked log2-fold gene expression changes in Pax5Jak2/+ (PJ) B-ALLs versus control (Ctrl) Pax5+/- Cdkn2ab+/- B-ALLs (left). NES, normalized enrichment score. The expression of five genes that are upregulated in Pax5Jak2/+ B-ALL cells (upper row) and Pax5-/- (KO) pro-B cells (lower row) is shown in (E). Likewise, the expression of five genes that are upregulated in control B-ALL cells (upper row) and Pax5+/+ (WT) pro-B cells (lower row) is shown in (G). TPM, transcripts per million. Mean TPM values with SEM are shown for the following RNA-seq experiments: 3 (WT pro-B), 2 (KO pro-B), 2 (Ctrl B-ALL), and 4 (PJ B-ALL). (H) Expression of the Ptprc (CD45) gene from exon 3 to exon 8, as determined by RNA-seq of Pax5Jak2/+ and Pax5+/- Cdkn2ab+/- B-ALL cells. The alternatively spliced exons 4 (A), 5 (B) and 6 (C) are indicated in orange in the respective exon-intron structure of the Ptprc gene. Ptprc transcripts of B220+ Pax5+/- Cdkn2ab+/- B-ALL cells contain all three exons, thus giving rise to expression of the CD45 isoform RABC (known as B220). In contrast, reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2/+ B-ALL cells, which likely gives rise to the CD45 isoforms RBC and RB
Figure 3A. Differentially expressed genes (DEGs) analysis based on RNA-seq data. Compared to the Control-LNA L2C, 723 genes were up-regulated and 521 genes were down-regulated in LincGET depleted embryos. B. KEGG pathway analysis of DEGs showed that the MAPK signaling pathway is mainly affected by LincGET depletion. C. The expression of key factors in the ERK1/2-MAPK or LNK/P38-MAPK signaling pathways was significantly affected by LincGET depletion. Embryos injected with LNA were collected at phCG 48 hours at late 2-cell stage for SG-qPCR analysis. The error bars represent s.e.m. About 150 embryos were used for each group, and three experimental replicates were performed. Two tailed student's t-test was used for statistical analysis. D. Western blot analysis indicates that the protein and phosphorylation level of p38 and ERK1/2, key kinases in the MAPK signaling pathway, decreased in LincGET-depleted 2C. Embryos injected with LNA were collected at phCG 48 hour at late 2-cell stage for western blot analysis and about 200 embryos were used for each lane. Three experimental replicates were performed. E. Immunofluorescence indicates that the phosphorylation level of P38 and ERK1/2 decreased dramatically in LincGET-depleted 2C. Embryos injected with LNA were collected at phCG 48 hours at late 2-cell stage for IF analysis. Scale bar, 50 μm. Three experimental replicates were performed and about 15 embryos were used in each group.F. Quantification of the fluorescence intensity indicates that the phosphorylation level of P38 and ERK1/2 decreased dramatically in LincGET-depleted 2C. Two tailed student's t-test was used for statistical analysis. Related to Figure 3E and Appendix Fig S4B. G. The expression of some target genes of the ERK1/2-MAPK or LNK/P38-MAPK signaling pathways was significantly affected by LincGET depletion. Embryos injected with LNA were collected at phCG 48 hours at late 2-cell stage for SG-qPCR analysis. The error bars represent s.e.m. About 150 embryos were used for each group, and three experimental replicates were performed. Two tailed student's t-test was used for statistical analysis.
a) Reduced levels of soluble mTOR (as a function of tubulin) in stable inducible PC12 cells expressing mutant (Gln74; Q74) huntingtin compared with those expressing wild-type (Gln23; Q23) huntingtin (four independent experiments; P 0.figf2(a) Reduced levels of soluble mTOR (as a function of tubulin) in stable inducible PC12 cells expressing mutant (Gln74; Q74) huntingtin compared with those expressing wild-type (Gln23; Q23) huntingtin (four independent experiments; P 0.0001; unpaired t-test). (b) Reduced levels of soluble mTOR in brain lysates from mice expressing mutant huntingtin (M1, M2; n = 9) compared with age-matched wild-type littermates (C1, C2; n = 9; P 0.001, unpaired t-test). (c) COS-7 cells transiently transfected with wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) labeled for endogenous 4E-BP1 (red). (d) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease showed 4E-BP1 in intranuclear inclusions. No 4E-BP1-stained inclusions were seen in controls (data not shown). ( (e) Western blot of cell lysates from stable inducible PC12 cells transfected with wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin shows that cells expressing mutant huntingtin had 31% (± 4.3% s.e.) less phosphorylated S6K1 (top) relative to total S6K1 (bottom) than cells expressing wild-type huntingtin. (f) Similarly, cells expressing mutant (Gln74; Q74) huntingtin had 30% (± 4.5% s.d.) less phosphorylated 4E-BP1 (top) relative to total 4E-BP1 (bottom) than cells expressing wild-type (Gln23; Q23) huntingtin. Expression of wild-type and mutant huntingtin was induced for 48 h (e,f). (g) Cells expressing mutant (Gln74; Q74) huntingtin also had lower levels of phosphorylated forms of transiently transfected FLAG-tagged 4E-BP1 than cells expressing wild-type (Gln23; Q23) huntingtin. The multiple bands in 4E-BP1 (f,g) are due to its different phosphorylated species.
Figure 8At T=0 hormone-repressed genes are active with RNA pol II and an open chromatin configuration decorated by acetylated histones and FOXA1. Upon hormone exposure the HP1γ-LSD1.com complex interact with the ATPase BRG1 and is actively recruited to the target genes along with the kinases ERK, MSK1 and CDK2, responsible for PR phosphorylation in S294 and S400. Once bound to the chromatin the complex promotes histone deacetylation, demethylation and chromatin remodeling via BRG1, which increase nucleosome positioning and occupancy. This arrangement of nucleosomes constitutes a better platform for linker histone H1.2 binding, and thus close the target chromatin decreasing RNApol II loading and transcription. The factors involved in hormone-dependent repressive complex anchoring to target genomic regions as 1) PR-Hormone Responsive Element DNA binding, 2) FOXA1-BRG1 interaction and 3) HP1γ-H3K9me3 binding are highlighted.
Figure 1A. Domain structure of human WDTC1 with locations of the H-box and WDXR motifs indicated. Alignment of WDTC1 H-box motifs in different species (boxed in grey) and bolded orange represents key residues contacting DDB1 [13]. Alignment of DWD boxes in different DWD proteins (bottom right) with WDXR submotif indicated; tandem DWD boxes are shown for WDTC1 only. B. Endogenous CUL4A and CUL4B complexes were isolated from 293T cell lysates by immunoprecipitation (IP) and associated proteins were detected by immunoblotting (IB). Mock includes beads only control. C. Flag-tagged WT and mutant WDTC1proteins were transiently expressed in 293T cells. Flag-WDTC1 complexes were immunoprecipitated with anti-FLAG and their associated proteins were detected by immunoblotting as indicated; EV, empty vector control. D. Top, the modeled structure of WDTC1 in complex with CRL4 (PDB 4A0K); WDTC1 superimposition was based on its H-box (PDB 317N). WDTC1 domains are rendered in same colors as linear domain structure in A; WDXR arginines were rendered in dark blue ball-and-sticks. Bottom, schematic summarizing results presented in C. E. 293T cells were transfected with HA-ubiquitin along with various combinations of plasmid and siRNA as indicated. Transfected cells were lysed under denaturing conditions to obtain whole cell extracts (WCE) and immunoprecipitated with anti-FLAG. WDTC1 ubiquitylation was evaluated by immunoblotting as indicated. F. 293T cells transiently expressing Flag-WDTC1 were transfected with scramble (scrm) siRNA or siRNAs against DDB1, CUL4A and CUL4B. Flag-WDTC1 levels and knockdown efficiency were assessed by immunoblotting as indicated.
Figure 3A, B) HepG2 cells were treated for the indicated time with 30 µM 3,4-DC (A) or indicated dose of 3,4-DC for 8 h (B). Thereafter, cells were collected and SDS-PAGE and immunoblots were performed as described before. Total p70S6K, phosphorylated p70S6K at T389 (P-p70S6K), Atg14, Lamp1, LC3, p62, and GAPDH protein levels were measured with specific antibodies. Samples for immunoblots in A and B were run in parallel instances, then cut into horizontal stripes and probed separately. Blots probed for P-p70S6K were stripped and reprobed to detect p70 independently from its phosphorylation status. The same applies for LAMP1 and GAPDH. (C, D) H4-GFP-LC3 cells were treated with 30 µM 3,4-DC in the presence or absence of CHX or AMD or with CQ for 16 h as controls, as indicated. GFP-LC3 dots were quantified in (D). Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus untreated control; # = p < 0.05 versus untreated with CQ). (E) RNA expression levels 16 h after exposure to 3,4-DC of the indicated genes in HepG2 cells were measured by RT-PCR and normalized to the expression of the housekeeping gene (GAPDH). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001). ,G) U2OS GFP-LC3 cells were enucleated to obtain cytoplasts, which were further treated with 3,4-DC and torin1. GFP-LC3 dots in cytoplasts were counted as shown in (G). Data are means ± SD (** = p < 0.01). Representative images with cytoplast marked by a white square are shown in (F). Scale bar equals 10 µm.
Atg11 and Atg8 bind Atg36.Figure 7(A) Coimmunoprecipitation of Atg11 and Atg8 with Atg36. IP was performed in either C13-Atg36-PtA atg1Δ or C13-Mvp1-PtA atg1Δ cells containing plasmids expressing either HA-Atg11 or HA-Atg8 under control of the TPI1 promoter. Cells were grown for 24 h to post‐log phase in glucose medium followed by growth in oleate medium for a further 18 h. Cells were then grown under non‐starvation or starvation conditions for a further 2 h. IgG Sepharose beads were used to immobilise Atg36-PtA or Mvp1-PtA from spheroplast yeast lysates. After extensive washing, bound material was eluted by Tev protease cleavage. SDS-PAGE gels (percentage indicated) were probed with anti‐HA (monoclonal 12CA5) and PAP. TL, total lysate; IP, immunoprecipitate (Tev eluate). (B-D) GFP-Atg11 was expressed in strains as indicated and cells were grown to post‐log phase in glucose medium. Inset shows magnification plus individual slices of stack from atg1Δ cells. Bar, 5 μm.
Figure 4(A and B) FUS disrupts Ca2+ homeostasis. HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K as indicated. Release of ERCa2+ was induced by treatment of cells with OxoM. (A) shows cytosolicCa2+ levels with representative Fluo4 fluorescence traces on the left and normalized peak values on the right. Fluo4 fluorescence shows a transient increase in cytosolicCa2+ levels upon OxoM treatment but compared to control, wild-type and mutant FUS all increase peak cytosolicCa2+ levels. (B) shows mitochondrialCa2+ levels with representative Rhod2 fluorescence traces on the left and normalized peak values on the right. Data were analysed by one-way ANOVA and Tukey's post hoc test. (A) N=49-52 cells from 3 experiments; (B) N=50-52 cells from 5 experiments, error bars are s.e.m.; *p<0.05, **p<0.01, ***p<0.001. (C) FUS reduces mitochondrial ATP production. ATP levels were measured in NSC34 cells transfected with the ATP indicator AT1.03 and either control vector (CTRL), HA-FUS, HA-FUSR521C or HA-FUSR518K. Cells were imaged in time-lapse prior to and after KCN treatment to inhibit oxidative phosphorylation. Representative traces of YFP/CFP ratios are shown for the different samples; initial YFP/CFP ratios prior to KCN treatment and those after KCN treatment are indicated. The fall in YFP/CFP ratios correlates with ATP produced by oxidative phosphorylation. Bar chart shows relative ATP levels produced by oxidative phosphorylation (OXPHOS) in the different samples.