EMBO/sd-character-level-ner huggingface dataset:
Figure 3Volcano plot of differentially expressed genes (DEGs) in DMSO- and R406-treated cells on d12. Red dots represent upregulated genes and blue dots represent downregulated genes in R406-treated cells. Dash lines represent 2-fold change, adjusted p-value < 0.05. Statistical significance was assessed by the method described previously (Love et al., 2014). Heatmap showing the normalized expression (z score) of DEGs. Red indicates up-regulated, whereas blue indicates down-regulated genes. KEGG analysis of upregulated genes in R406-treated reprogramming intermediates. Statistical significance was assessed by Benjamini-Hochberg method. Heatmap of RNA-seq data for the normalized expression (z score) of genes involved in glycine, serine and threonine metabolism. Red indicates up-regulated genes, whereas blue indicates down-regulated genes. RT-qPCR analysis of Gldc, Cbs and Tdh expression in R406- and shSyk-treated cells (E) and FK506-, shPpp3ca- and shNfatc1-treated cells (F) on d8. n = 3. Schematic of CUT&Tag experiments to map the genomic occupancy of NFATc1. NFAT motif enrichment and associated p-value are shown. Statistical significance was assessed by the method described previously (Heinz et al, 2010). NFATc1 occupancy at Gldc, Cbs and Tdh gene loci in cells treated with DMSO and R406 and no-antibody control. The predicted NFAT binding sites at the promoters of Gldc, Cbs and Tdh. qPCR assay of the binding of NFATc1 at Gldc, Cbs and Tdh gene loci in cells treated with DMSO and R406. n = 3.
TLN accumulation occurs in a compartment distinct from classic biosynthetic and endosomal pathways.Figure 4.TLN accumulation occurs in a compartment distinct from classic biosynthetic and endosomal pathways. (A-F) Early compartments. No colocalization was observed between TLN (green in all panels) and nuclei (A, TOPRO-3), ER-markers BIP (B) and calnexin (C), ERGIC-53 (D), β-COP (E) or GM130 (F), and labeling cis-Golgi. (G-L) Late compartments. TLN accumulations are not accessible for exogenous biotin (G). Early and recycling endosomes (H, EEA1) and transferrin receptor (I, TFR), late endosomes (J, LBPA), and lysosomes (K, Lamp-2; L, catD) also stained negative for TLN. Vertical sections (x-z, arrowheads in I and J) clearly distinguish TLN accumulations from recycling (I, TFR) and late endosomes (J, LBPA). Bars, 10 μm.
Wde controls the abundance and monoubiquitination level of Egg in the nucleus.Figure 3Immunofluorescence analysis of endogenous Egg (red) in OSCs using anti-Egg antibody before (siEGFP) and after (siWde) Wde depletion. DAPI (blue) shows the nuclei. Scale bar: 10 μm. Western blotting on fractionated materials of OSCs before (siEGFP) and after (siWde) Wde depletion. Anti-Egg, anti-Wde, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: whole OSC lysates. Immunofluorescence analysis of endogenous Egg (red) in OSCs upon Flag-Wde (green) co-expression. Anti-Egg and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm. Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Wde co-expression (green). Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm. Western blotting on fractionated materials of Myc-Egg-expressed OSCs before (-) and after Wde WT and Wde ∆CC co-expression. Anti-Myc, anti-Flag, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates. The schematic drawing of domain structures of Wde WT and its ∆CC mutant. The ∆CC mutant lacks the Egg-binding domain (842−907 a.a). Protein-protein interaction assays. Myc-Egg and Flag-Wde WT and its ∆CC mutant were expressed in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting. Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Flag-Wde WT and ∆CC mutant (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.
Figure 2Localization of CASPR in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelled with antibodies against CASPR1 and RIBEYE (A-B OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μm Localization o visual-arrestin (v-arrestin) in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelle with antibodies against v-arrestin and RIBEYE (C-D) V-arrestin, visual arrestin; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μm
Figure 5(A, B) MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and BRCA1 antibodies. Shown in (A) is the quantitation of a minimum of 100 EdU+ cells per replicate and the bars represent the mean ± S.D (n= 3 biological replicates). Representative micrographs are shown in (B). MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and RAD51 antibodies. Shown in (C) is the quantitation of minimum of 100 EdU+ cells per replicate and the bars represent the mean ± S.D (n= 3 biological replicates). MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and RAD51 antibodies. Representative micrographs are shown in (D). (E) Clonogenic survival assays using the indicated dose of olaparib and MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions. Data is shown as the mean ± S.D (n=3 biological replicates). Representative clonogenic images are shown in Figure EV5D.
Figure 6(A) Amino acid sequence alignment of HOIL-1, HOIP and Parkin centred at the start of the IBR domain. Conserved zinc coordinating residues are highlighted in yellow, other highly conserved residues are highlighted with dark blue, and weaker conservation of property is indicated by light blue highlight. Red triangles denote the position of the mutations indicated. (B) Time-course assay performed at 30°C featuring wild-type HOIL-1 in the absence or presence of the indicated ubiquitin oligomers, visualised by Coomassie staining. (C) HOIL-1[I358R], assayed as in (B). (D) HOIL-1[F381A/E383A], assayed as in (B). (E) The truncated HOIL-1[233-510; I358R] mutant was assayed as in (B). (F) The truncated HOIL-1[233-510; F381A/E383A] mutant was assayed as in (B). (G) The sequence from RING1-IBR was aligned by overlaying the HOIL-1 AlphaFold prediction (residues 282-404, AlphaFold Uniprot ID: Q9BYM8) with residues 699-827 in the crystal structure of HOIP-RBR (PDB: 5EDV). The location of the allosteric ubiquitin from that HOIP structure is also shown. HOIL-1 residues I358, F381 and E383 are highlighted in red. (H) Full-length HOIL-1 AlphaFold structural prediction (AlphaFold Uniprot ID: Q9BYM8) with likely location of allosteric ubiquitin shown. The region shown in (G) is indicated.
Figure 4A Representative immunohistochemical images of S1P, ETFA and ETFB in normal controls and patient 1. Scale bars: 100 μm. B Unchanged mRNA levels and decreased protein levels of ETFA and ETFB were observed in the skin biopsy of patient 1. Quantitative RT-PCR analysis (upper panel, n = 6 biological replicates of normal controls; n = 6 technical replicates of patient 1) and immunohistochemistry (lower panel, n = 6 biological replicates; n = 3 technical replicates) of the S1P, ETFA and ETFB levels. C Representative immunohistochemistry images of S1P, ETFA and ETFB in both Mbtps1-conditional knockout (cKO) and Mbtps1-loxP mice. Scale bars: 100 μm. D MBTPS1 gene knockout led to decreases in the ETFA and ETFB protein levels in vivo. Quantitative RT-PCR analysis (upper panel, n = 6 biological replicates) and immunohistochemistry (lower panel, n = 6 biological replicates) of the S1P, ETFA and ETFB levels in Mbtps1-cKO mice and Mbtps1-loxP mice. E Cycloheximide (CHX) chase analysis showed that MBTPS1 knockout induced rapid degradation of ETFA and ETFB proteins in HaCaT cells. Left panel: Representative western blotting images of the ETFA and ETFB protein levels during CHX chase. Right panel: Quantification of the immunoblotting results corresponding to the left panel (n = 3 biological replicates). F Mutant S1P (p.Val355Gly and p.Ter1053Arg) only weakly interacted with ETFA and ETFB. We constructed Flag-tagged wild-type and mutant S1P HaCaT cell lines and then performed a Co-IP assay with Flag antibody, and the interaction between S1P and ETFA/ETFB was detected by immunoblotting. G Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), decreased the association between ETFA and ETFB. We performed a Co-IP assay with an ETFA antibody, and the interaction between ETFA and ETFB was detected by immunoblotting. H Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), enhanced the incorporation of FAD into the ETF complex. The visible spectra of flavin show two shoulder peaks at 420 nm and 460 nm, indicating the incorporation of FAD in the ETF complex. In the presence of the wild-type S1P protein, the two peaks were shifted by 0.02 OD units, whereas the additional mutant S1P (p.Val355Gly and p.Ter1053Arg) weakly shifted the two peaks by 0.005-0.01 OD units.
Serum from a convalescent SARS patient cross-neutralizes 2019-nCoV-S-driven entry.Fig. 4.entry.Pseudotypes harboring the indicated viral surface proteins were incubated with different dilutions of serum from a convalescent SARS patient and subsequently inoculated onto 293T cells that transiently express ACE2 in order to evaluate cross-neutralization. The results from a representative experiment with triplicate samples are shown and were confirmed in a separate experiment. Error bars indicate SD. Statistical significance was tested by two-way ANOVA with Dunnett posttest.
Figure 7(A) VEGFR2 protein levels in wild-type (WT) and EVL-deficient (EVL-/-) endothelial cells. Actin was used as loading control. The lower panel shows quantification of VEGFR2 to actin ratios from five independent cell batches; error bars represent SEM; n.s. non-significant (p=0.388), unpaired Student's t-test. (B) Antibody feeding assay to monitor VEGFR2 internalization after VEGF stimulation. Left panel: schematic diagram showing the two step staining procedure of surface and internalized VEGFR2 antibodies, which are displayed in yellow and green in the merged confocal images of WT and EVL-/- endothelial cells (middle panel), respectively; scale bar, 10 µm. The bar graphs show quantification of the number of VEGFR2 clusters and the ratio of internalized to total VEGFR2 ratios normalized to wild-type; n = 4-6 independent cell batches, error bars represent SEM; **p<0.01, ***p<0.001, unpaired Student's t-test.
Figure 9PPM: parasite plasma membrane, IMC: inner membrane complex, and SPM: subpellicular microtubules.
iNPH-associated deletions disrupt the ability of Cwh43 to regulate the localization of GPI-anchored receptors.Figure 1A. Sanger DNA sequencing data from two iNPH patients confirming the presence of heterozygous CWH43 deletions. Arrows identify location of the deletion. B. Diagram of the domain structure of the Cwh43 protein illustrating the location of the damaging CWH43 deletions. Lower panel details the disruptive effect of the k696Asnfs mutation on the C-terminal endoplasmic reticulum export signal (YF), replacing it via a frameshift with a 23 amino acid sequence that contains an endoplasmic reticulum retention signal (KKKS). C. Fluorescence micrographs of HeLa cells transfected with plasmids encoding human GFP-Cwh43, GFP-Cwh43-k696fs or GFP-Cwh43-Leu533ter fusion proteins. To label the ER, HeLa cells were transduced with a baculovirus encoding a fluorescently labeled RFP-calreticulin fusion protein containing an ER retention signal (RFP-calreticulin-KDEL). To label the Golgi apparatus, cells were immunostained for golgin-97 (red). When compared with wild type GFP-Cwh43, GFP-Cwh43-k696fs and GFP-Cwh43-Leu533 showed decreased association with the Golgi apparatus (but not the ER) and were diffusely distributed throughout the cytoplasm. Scale bar is approximately 5 µm. D. Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type HeLa cells and two independent CRISPR CWH43 knockout (KO) HeLa cell lines in which a mutated CWH43 gene encodes a protein that is truncated near Leu533 and CWH43 mRNA and protein are markedly reduced. Cells were transfected to overexpress a control GFP plasmid, a plasmid encoding human wild type Cwh43 with GFP fused to the N-terminus, or a plasmid encoding human CWH43 harboring the iNPH-associated mutation (Lys696AsnfsTer23) with GFP fused to the N-terminus. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.
Figure 3(A) Primary sequences of the WT peptide (red) and P182L peptide (blue). The IxI/V motif is indicated with white text. Below: regions of 2D 1H-15N HSQC spectra of 15N-labeled cHSP27 (black) in the presence of increasing amounts of WT (red) or P182L peptide (blue). Resonances in the β4 and β8 strands broaden and disappear upon peptide binding, indicative of intermediate exchange. (B) Zoomed-in regions of resonances during the peptide titrations. The color bars indicate the amount of added WT or P182L peptide. (C) The combined 1HN and 15N chemical shift changes, denoted as chemical shift perturbation (CSP), are plotted as a function of added peptide concentration. (D) CSPs shown as a function of residue number for the WT (red) and P182L peptide (blue). (E) X-ray structure of cHSP27 bound to the WT peptide (PDB: 4mjh), shown in white sticks, with the results from (D) plotted onto the structure.
Figure 3Time-lapse confocal images of wild-type AX3 cells expressing tPH-CynA-KikGR (green) and PH-crac (red) before and after PI3K inhibition by 5 µM Wortmannin. Kymographs of PH-Crac intensity on the perimeter of one cell undergoing random migration in (A). White dotted line shows addition of Wortmannin. Intensity plot across the white dotted line in tPH-CynA-KikGR expressing cell before and after Wortmannin treatment in (A). Confocal images of tPH-CynA-KikGR expressing wild-type Dictyostelium AX3 cells before (left) and after (right) LY2924002 treatment. Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wildtype Dictyostelium AX3 cells before and after LY2924002 treatment, T-test was carried out by GraphPad Prism, **P ≤ 0.01 versus -LY2924002 group (mean±SEM, n = 7). Confocal images of tPH-CynA-KikGR (green) and PH-Crac (red) expressing wild-type Dictyostelium AX3 cells (left) and PI3K1-2- Dictyostelium cells (right) treated with 5 µM Latrunculin A. Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wildtype Ax3 and PI3K1-2- cells. There was no statistically significant difference of the biosensor on the membrane between the two groups (mean±SEM, n = 17). Confocal images of tPH-CynA-KikGR (green) and LimE (labeling F-actin) expressing PI3K1-2- cells. Front and back of the cell are shown and white arrow showing the back localization. Confocal images of tPH-CynA-KikGR (green) expressing PI3K1-2- cells, white box showing the vesicle, and white arrow showing the back localization.
a) Primary cortical neurons were transduced with lentivirus.f8(a) Primary cortical neurons were transduced with lentivirus expressing sh-RNA for Nrf2 or rat NDP52 (rNDP52), or a scrambled sh-RNA at DIV 1, and were maintained until DIV 6. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using 12E8- and PHF1-specific antibodies, respectively. Total tau was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot. (b) Bar graph of the relative optical density of phosphorylated tau normalized to actin. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; ***P0.001) (c,d) Primary cortical neurons were transduced with a control lentivirus (FIGB) or with one expressing humanNDP52 (hNDP52) at DIV 1. To induce autophagy, trehalose (150 mM) was added at DIV 5 and the neurons incubated for 24 h (DIV 6). Primary cortical neurons were fixed with 4% paraformaldehyde, and stained with the 12E8 or PHF1 antibodies. Scale bar, 20 μm. The optical density of tau phosphorylated at Ser262/Ser356 (12E8). (e) and Ser396/Ser404 (PHF1) (f) in the soma of ~30 neurons randomly chosen was analysed with the ImageJ program. Data were analysed using Student's t-test (***P0.001).
AVs in COPI-depleted cells are not degradative.Figure 6.AVs in COPI-depleted cells are not degradative. (A) Long-lived protein degradation was assessed 72 h after transfection with control or β′-COP siRNA in 293/GFP-LC3 cells. Cells were either starved of amino acids for 2 h (St) or incubated in fresh growth medium (Fed) for 2 h. The amount of protein degradation is presented as mean ± SEM. (B) siControl-treated cells and β′- and α-COP were depleted for 48 h, then incubated with Lysotracker red (50 nM) for 30 min before fixation. Merged image shows very little colocalization of GFP-LC3 AV-positive structures. Asterisk shows cell enlarged in inset (C) After β′- and α-COP siRNA depletion, Magic Red-RR2 (MR) was used to detect active cathepsin B in cells which were fed or starved (St) for 2 h. The intensity of MR labeling was measured in live cells by flow cytometry.
Nucleoside degradation causes S7P accumulation due to depletion of the other transaldolase substrate glyceraldehyde-3-phosphate, and SBP accumulation due to phosphorylation of S7P into SBP by Pfk1.Figure 6(A) Diagram of proposed reactions. (B‐E) DHAP, SBP and S7P levels in wild type, tkl1/tkl2 (B), tal1/nqm1 (C) and pfk1 (E) strains upon carbon starvation, and wild‐type strain upon abruptly switching from glucose to no carbon (WT) versus to dihydroxyacetone (WT+DHA) as the sole carbon source (D). The x axis represents hours after carbon starvation, and the logarithmic y axis represents absolute intracellular concentration (mean±range of N=2 biological replicates).
Figure 1Fate of Purkinje cells in mice with Purkinje-cell-specific knockout of Ccp1. Ccp1flox/flox L7-cre mice show a massive but incomplete degeneration of Purkinje cells (calbindin-stained; red) at 4 months, while all Purkinje cells are degenerated at 18 months. Scale bar: 500 µm. Schematic representation of enzymatic tubulin polyglutamylation (red points are glutamate residues that are posttranslationally added to C-terminal tails of α- and β-tubulin). In neurons, TTLL1 is generating a large share of the overall polyglutamylation (Janke et al, 2005), and CCP1 is reversing this PTM. Loss of CCP1 induces TTLL1-mediated hyperglutamylation, which can be avoided by TTLL1 inactivation. Fate of Purkinje cells in mice with combinatorial knockout alleles. Knockout of Ccp1 (pcd-mouse; Ccp1-/-) results in a complete degeneration of Purkinje cells (calbindin-stained; red), while the additional knockout of Ttll1 selectively in the Purkinje cells (L7-cre) protects the entire Purkinje cell layer from degeneration up to 18 months. Scale bar: 500 µm. Nissl staining of the Purkinje cell layer of the brains from various knockout mice shown in (C) confirms the absence of Purkinje cells in Ccp1-/-, and the presence of a wild-type-like Purkinje-cell density in Ccp1-/- Ttll1flox/flox L7-cre mice. Scale bar: 20 µm. Purkinje cell layer (PC) and granule cell layer (GC) are indicated. Immunoblot of tissue extracts from cerebella of Ttll1-/- and wild type mice, probed with the polyE, anti-∆2-tubulin and 12G10 antibodies. Increasing amounts of extracts were loaded for detection of polyglutamylated proteins with polyE. Schematic representation of the experimental paradigm applied in this figure. The thick line in the cerebella symbolises the Purkinje cell layer.
Schematic of experimental workflow and top 50 sorted β-catenin interactors shown with GO cellular component analysis.Figure 1Workflow for identification of the β-catenin interactome in A10 cells The list of potential β-catenin interacting partners. β-catenin (blue), is present in all replicates. Previously reported cytoplasmic and nuclear interacting partners, α-catenin and Mediator 12, are highlighted in yellow and green, respectively. FXR1, which shares a high sequence and domain homology with FMRP, is highlighted in purple A first level Gene Ontology (GO) domain analysis of the top 50 candidates in the GO domain 'cellular component'. The percentage of proteins associated with each GO term of the respective domain is represented in the pie chart
Protein correlation profiling (PCP) analysis of glycerol density gradient-separated proteasome complexes.Figure 1PCP-MS strategy to identify proteins interacting with specific proteasome subtypes. U937 cells were cross-linked with formaldehyde and lysed, and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device. Protein complexes were then separated on a 15-40% glycerol gradient. Each fraction of the gradient was analyzed by nano-LC-MS/MS. Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein, after internal standard calibration using a mix of 8 isotopically labeled peptides. The PCP analysis was performed as described in the Materials and Methods section. PCP analysis of the 19S regulatory complex. Protein abundance profiles of 16 proteins of the 19S RP (Rpt1-6, Rpn1-3, Rpn5, 7-9, 11-13, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values (representing the Euclidian distance between the abundance profile of each protein and the reference profile) of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits (right panel). PCP analysis of proteasome 20S complex. Protein abundance profiles of 17 proteins of the 20S CP (α1-α7, β1-β7, β1i, β2i, β5i, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits.