EMBO/sd-character-level-ner huggingface dataset:
The nonfusogenic SipB428–593 mutant localizes to the mitochondria, but does not form multivesicular structures and interferes with the activity of the wild-type protein.Figure 5. The nonfusogenic SipB428-593 mutant localizes to the mitochondria, but does not form multivesicular structures and interferes with the activity of the wild-type protein. (A) COS-2 cells were transfected with a plasmid expressing YFP-tagged SipB428-593, stained with an antibody to the mitochondrial protein CoxIV, subunit VIc (Molecular Probes, Inc.), and examined by confocal microscopy. SipB428-593-YFP localized to mitochondria and did not induce the formation of multivesicular structures seen after expression of the wild-type protein.  (B) COS-2 cells were cotransfected with plasmids expressing either SipB428-593-YFP or SipB-CFP, and transfected cells were examined under a confocal microscope. Expression of SipB428-593-YFP prevented wild-type SipB-CFP from forming multivesicular structures (notice that vesicular structures are only apparent in the absence, but not in the presence, of SipB428-593-YFP; right).
Figure 5The putative SRSF2 binding motifs on NUMB exon 9 (labeled as red), predicted by RBPmap. M1 refers to motif 1 and M2 refers to motif 2. Western blotting showing the overexpression of SRSF2 in 293T cells, which was co-transfected with NUMB reporters of WT, ∆M1 or ∆M2. ∆M1 or ∆M2 represents plasmid deleting corresponding binding site. A minigene splicing reporter assay showing SRSF2 promoted the generation of NUMB_L isoform via M1 but not M2. The upper panel indicating the pET01 vector that contains two constitutive NUMB exons. The representative RT-PCR electropherogram showing the expression of NUMB_L and NUMB_S isoforms in WT, ∆M1 or ∆M2 transfected 293T cells with or without SRSF2 overexpression, respectively. The bar graph showing the normalized exon 9 inclusion from the RT-PCR electropherogram of (C) to the WT control cells. P-values were determined by an unpaired two-tailed Student's t-test. On day 5 of hematopoietic differentiation, the inclusion or exclusion of NUMB exon 9 were detected by RT-PCR with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5. The bar graph showing the signaling intensity of each specific band from the RT-PCR electropherogram. The top panel depicts the location of specific primers targeting NUMB_S and NUMB_L isoform. The bottom bar graph showing the expression of NUMB_S and NUMB_L in cells on day 5 of differentiation with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5 with RT-qPCR.
Figure 8(A). The 2Fo-Fc annealing omit map (contoured at 1.0σ) around MUNC18-1-pY145 phospho-segment. (B). Comparison of residues of PTP-MEG2 interacting with NSF and MUNC18-1. Amino acid residues of NSF and MUNC18-1 are coloured as follows: green, residues interacting with both NSF and MUNC18-1; red, residues specifically contributing to NSF recognition; blue, residues selectively contributing to MUNC18-1 interaction. (C). The structural alteration of the interactions surrounding Y471 of PTP-MEG2 with the MUNC18-1-pY145 site. (D). The structural alteration of the interactions surrounding I519 of PTP-MEG2 with the MUNC18-1-pY145 site. (E). Relative phosphatase activities of different PTP-MEG2 mutants towards the MUNC18-1-pY145 phospho-segment compared with wild-type PTP-MEG2. (F). Relative phosphatase activities of different PTP-MEG2 mutants towards the DYNAMIN 2-pY125 phospho-segment compared with wild-type PTP-MEG2.Data information: (E, F) Residues are coloured according to Figure 8B. (E, F), * P<0.05, ** P<0.01, *** P<0.001: PTP-MEG2 mutants compared with the control. N.S. means no significant difference. Data were obtained from 3 independent experiments. **, P<0.01; *** P<0.001 and ## P<0.01; ### P<0.001. N.S. means no significant difference. Data were obtained from 10 cells in each group of 6 independent experiments. . All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m. (G).The percentages of PSF and SAF for PTP-MEG2-R409A or R410A were calculated. Data information: (G), * indicates PTP-MEG2-R409A or R410A mutants overexpression group compared with the WT overexpression group. # indicates the PTP-MEG2 overexpression group compared with the control group. **, P<0.01; *** P<0.001 and ## P<0.01; ### P<0.001. N.S. means no significant difference. Data were obtained from 10 cells in each group of 6 independent experiments. . All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m. (H). Schematic illustration of the PTP-MEG2-regulated processes of vesicle fusion and secretion in chromaffin cells via the dephosphorylation of different substrates with distinct structural basis. PTP-MEG2 regulates vesicle fusion and vesicle size during the catecholamine secretion of adrenal gland by modulating the phosphorylation state of the pY83 site of NSF, which relies on the key residues G334, D335 (pY loop), Y471 (WPD loop), I519 (P-loop) and Q559 (Q loop). PTP-MEG2 regulates the fusion pore initiation and expansion procedures of catecholamine secretion by the adrenal gland (also designated as foot probability) by modulating the newly identified substrate MUNC18-1 at its pY145 site and DYNAMIN2 at its pY125, through distinct structural basis from that of its regulation of NSF phosphorylation.
Fig.Figure 1RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h, Ca WT or Ca YL, and Lm WT or Lm Δhly for 60 min. (A) Levels of Rab8A and Rab8A pT72 phosphorylation were analysed by Western blot and quantified by densitometry. Data represent the mean ± SEM of three independent biological experiments. One-way ANOVA followed by Dunnett's test compared to WT strains. ns= non-significant; *p ≤0.05; **p ≤0.01. (B) Recruitment of endogenous LRRK2 and EGFP-Rab8A to M. tuberculosis (Mtb) was visualised by confocal microscopy. Scale bar = 5 μm. (C) C. albicans (Ca) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates. (D) L. monocytogenes (Lm) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates.
The formation of the immunological synapse does not predict target outcome during CTL-tumor cell contactsLifeAct-GFP OT-I CTLs were cultured with B lymphoma cells expressing the OVA model antigen and the genetically-encoded calcium indicator Twich2B.Figure 3(A) Representative time-lapse image illustrating CTL rounding upon target engagement. Scale bar, 5 µm. (B) Quantification of CTL shape (roundness coefficient) in unconjugated and conjugated CTLs. A circular shape corresponds to an index of 1. Each dot represents one CTL. Bars represent mean values. ( Data information: Data were pooled from three independent experiments. (***p<0.001, ns, not significant, unpaired t‑test). (C) Representative time-lapse images showing examples of CTL-tumor conjugates resulting in either no detectable response, transient calcium response or sustained calcium response in the target cell. Arrows show actin accumulation at the immunological synapse. Scale bar, 5 µm. Data information: Data were pooled from three independent experiments. (***p<0.001, ns, not significant, unpaired t‑test). (D) Quantification of CTL roundness for contacts in the three indicated categories. Each dot represents one CTL. Red bars represent mean values. Data information: Data were pooled from three independent experiments. (***p<0.001, ns, not significant, unpaired t‑test). (E) Quantification of actin accumulation at the immunological synapse for CTLs engaged in contacts with the indicated outcome. Each dot represents one CTL. Red bars represent mean values. Data information: Data were pooled from three independent experiments. (***p<0.001, ns, not significant, unpaired t‑test).
Modeling human male infertility in mice using the autosomal knockin approach.Figure 5Schematic representation of missense and nonsense mutations in human TEX11 found exclusively in men with azoospermia. The full-length TEX11 protein (GenBank accession number: NP_112566) contains 925 residues. The tetratricopeptide-like (TPR-like) helical domain, found in proteins that form large complexes, extends from residues 161 through 499 (Blatch Lassle, 1999). Four of the residues mutated in azoospermic men (W117, V142, Q172, and V748) are conserved between human and mouse TEX11proteins (Supplementary Fig S1). Generation of three lines of Tex11 knockin mice. Each harbors a single amino acid substitution in TEX11 (indicated by asterisks) analogous to a missense mutation identified in a human patient. Nucleotide and respective amino acid changes are indicated. Exons 1, 2, and 3′UTR are from the mouse Tex19.1 gene.
Kinetic heterogeneity of the transport domain dynamics.Figure 4(A) Dwell-time distributions of the OFS (blue) and the IFS (red) for WT (top) and K290A (bottom) GltPh observed under equilibrium conditions in saturating Na+/l-Asp (left). Lines are fits to three exponentials. Data are averages and standard errors of at least three independent measurements. (B) Representative EFRET trajectories of WT GltPh showing different transition frequencies. Raw data are in black, and idealizations are in brown. Scalebar is above the trajectories. (C) Distribution of the WT (top) and the K290A GltPh (bottom) molecules with different mean transition frequencies (white bars). N is the number of trajectories longer than 90 s used in the analysis. The stacked black bars are fractions of trajectories without transitions. Pink bars show the expected binomial distribution if all trajectories shared the mean transition frequency of 0.04 s-1 for the WT and 0.34 s-1 for the K290A. Data are averages and standard errors of at least three independent measurements. (D) 2D-histograms of the consecutive dwell lengths in the OFS (top) and the IFS (bottom). From left to right: calculated distribution of dwell times randomly selected from the distributions in panel A; measured distribution for WT GltPh and the K290A mutant. (E) Representative trajectories of the WT (left) and the K290A (right) GltPh molecules showing switching between dynamic modes. Raw data are in black and idealizations are in brown (top). Black and grey bars under the trajectories indicate apparent slower and faster dynamic modes. Survival plots for the OFS and the IFS (middle). Solid lines are fits to a single (IFS) and double (OFS) exponentials. Dashed lines (OFS only) are rejected fits to single exponentials. Autocorrelation plots of the sequential dwell durations (bottom).
Fig2.Figure 2Simulated fluxes of amino acids compared with experimentally measured data. For each amino acid, simulated fluxes of uptake and tRNA charging are displayed together with the measured uptake flux. The upper bound on the uptake is also displayed, which is the linear trendline of the experimentally measured data if the amino acid is consumed rather than being secreted. The measured data are from the study (Goel et al, 2015). , Scaled reduced cost analysis for uptake rates of amino acids at different growth rates. Colour represents the scaled reduced cost value. A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate.
Figure 3A-B, Single-molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse-labelled with IdU (10 min) and CIdU (20 min). (A) Distribution of inter-track distances. Bars represent the median. n > 100. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. (B) Size distribution of CldU track length. Bars represent the median. n > 300. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. C, Distribution of intertrack distances measured by IdU/CIdU pulse labelling and DNA fiber analysis. Bars represent the median. n > 110. Mann-Whitney: **** P < 10-4. One representative experiment out of two biological replicas is shown. D, Scatter plot of chromatin-bound GFP-RPA1 intensity per cell. GFP-RPA1 and RFP-PCNA reporter cells were transfected with indicated siRNAs and treated 2 hrs with UCN-01 (300 nM) prior to pre-extraction and fixation. GFP-RPA1 levels were measured in PCNA positive cells. Lines represent medians. n > 150. t-tests: **** P < 10-4, * P < 0.05; a.u., arbitrary unit. E, Analysis of replication factor loading. Soluble proteins from siRNA transfected cells treated as in D where removed by CSK-T extraction and chromatin pellets analysed by western blotting. One representative experiment out of three independent biological replicas is shown. F, Quantification of chromatin-bound MCM2 (left) and CDC45 (right) from the three independent experiments described in (E). MCM2 and CDC45 levels were normalized to histone H4 levels and shown relative to untreated control. t-tests: *** P < 10-3, ** P < 0.01, * P < 0.05, n.s. non-significant. G, Analysis of chromatin-bound proteins in HBO1 depleted cells as in E. One representative experiment out of two biological replicas is shown.
SCFFbxw5 regulates MCAK in G2/M A.Figure 6A. Cycloheximide (CHX) chase experiments of RPE-1 cells treated with the indicated siRNA for 48 hours and then either grown further asynchronously or arrested in S phase with 2 mM hydroxyurea (HU) or in G2 with 10 µM RO-3306 for 18 hours. 50 µg/ml CHX was added to the cells, samples were harvested at the indicated time points and analysed via SDS-PAGE and western blotting using the indicated antibodies for detection. Bottom: Representative blots. Asterisks indicate an unspecific band detected by the Fbxw5 antibody. Top: Quantification of MCAK/Uba2 signal intensity ratios normalised to time point zero. Error bars show standard deviation of 4 independent experiments and asterisks indicate p-value of a two-tailed unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). B. CHX chase experiment as in A, except that cells were treated with the indicated siRNAs for 48h and arrested with 200 mM thymidine for 16h, released for 9 hours and again arrested for 16h. Following the 2nd release, 50 µg/ml CHX was added as indicated and samples were analysed as in A. Left: representative blots. Asterisks indicate unspecific bands detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal intensity ratios normalised to time point zero. Error bars show standard deviation of 6 independent experiments and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01). C. Extracts of RPE-1 cells treated with the indicated siRNAs for 72 hours, grown asynchronously or arrested with the indicated compounds for the last 24 hours and treated either with DMSO (-) or 10 µM MG-132 (+) for the very last 6 hours. Left: Representative blots. Asterisk indicates an unspecific band detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal ratio normalised to non-targeting control with DMSO of 6 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test (**P < 0.01).
Counter-transported ion binding siteA.Figure 4A. Purified EAAT1CRYST takes up L-glutamate in liposomes loaded with K+ or Rb+, but not with choline (Ch+), or with Ch+ and Ca2+. Plots depict data as in Fig. 3A. B. Superimposition of Na+/transmitter and Rb+ bound EAAT1CRYST structures, respectively, with tranD depicted in orange (Na+/transmitter) and pink (Rb+). TM4 and UCPH101 are omitted for clarity. Black mesh depicts anomalous-difference map contoured at 3.5σ around the tranD. Rb+ coordination is shown in the inset. Residue numbering corresponds to EAAT1WT, and the coordination distance (angstrom) is parenthesis.
Figure 2IRE1α inhibition compensated MITOL-KO MEFs for vulnerability to ER stress. MEFs were transfected with scramble or siIRE1α (#1, #2). After 24 hours, these MEFs were exposed with Tu for 24 hours , followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3). MEFs were treated with 4µ8C (25 µM) for 2 hours prior to Tu treatment for 24 hours (B), followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3). MITOL KO did not affect the autophosphorylation of IRE1α. MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos-tag SDS-PAGE. MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. MEFs were transfected with either miR-17 or miR-34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours, followed by luciferase reporter assay (F). Error bars represent SD (n=3). MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR IRE1α-mediated JNK phosphorylation was prolonged by MITOL KO. MEFs were incubated with Tu for indicated periods and phosphorylated JNK was detected by immunoblotting with indicated antibodies. Error bars represent SD (n=3). JNK inhibition reduced ER stress-induced apoptosis of MITOL-KO MEFs. MEFs were treated with 20 µM JNK inhibitor-II (JNK inh) 2 hours prior to Tu treatment for 24 hours, followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).
p53 inhibition induces autophagy by ER stress (a–c) WT HCT116 cells were transiently transfected with GFP–LC3, treated with PFT-alpha for 2, 4 or 6 h and subjected to immunofluorescence staining to observe the colocalization of GFP–LC3+puncta with the ER marker ERp57 (a) or a mitochondrial marker (b).figf6p53 inhibition induces autophagy by ER stress (a-c) WT HCT116 cells were transiently transfected with GFP-LC3, treated with PFT-α for 2, 4 or 6 h and subjected to immunofluorescence staining to observe the colocalization of GFP-LC3+ puncta with the ER marker ERp57 (a) or a mitochondrial marker Complex III (b). Note that cells with GFP-LC3+ puncta often contain scarce amounts of ERp57 (white arrows) after prolonged incubation with PFT-α. The percentage of colocalization (means ± s.d., n = 3 experiments with 50 images per experiment) was determined by confocal microscopy (c). p53 inhibition induces autophagy by ER stress (a-c) WT HCT116 cells were transiently transfected with GFP-LC3, treated with PFT-α for 2, 4 or 6 h and subjected to immunofluorescence staining to observe the colocalization of GFP-LC3+ puncta with the ER marker ERp57 (a) or a mitochondrial marker Complex III (b). Note that cells with GFP-LC3+ puncta often contain scarce amounts of ERp57 (white arrows) after prolonged incubation with PFT-α. The percentage of colocalization (means ± s.d., n = 3 experiments with 50 images per experiment) was determined by confocal microscopy (c). (d) eIF2α phosphorylation in WT or p53−/− HCT116 cells treated with PFT-α (n = 5). (e, f) WT HCT116 cells (e, f) or p53−/− HCT116 cells (f) were transfected with GFP-LC3, plus a control siRNA or the indicated combination of IRE1α- and/or p53-specific siRNA, and treated with PFT-α, thapsigargin, tunicamycin or brefeldin A. (g) GFP-LC3-transfected WT or ire1α−/− MEF were treated with PFT-α. Columns in e-g show means ± s.d. of 3 independent experiments (*P 0.05).
SGK1 in skeletal muscle is critical for preservation of skeletal muscle.Figure 3A. Western blot analyses and densitometry of quadriceps muscles from non−hibernating and hibernating squirrels demonstrates significant increases in total SGK1 and phosphorylated SGK1. Immunostaining of quadriceps muscle for total SGK1 shows upregulation during hibernation. B. Levels of phosphorylated Foxo3a in S315 and T32 are significantly increased during hibernation. Densitometric analysis of P‐Foxo3a as a function of Foxo3a.
Lactobacillus acidophilus NCK2187 promotes intestinal regulation in steady state A B6 mice were orally gavaged with 109 CFU NCK56 (blue) or NCK2187 (green) on days 0, 3, 6, and 9, or left untreated, and immune responses in the colon analyzed at day 14 by flow cytometry.Figure 2A B6 mice were orally gavaged with 109CFU NCK56 (blue) or NCK2187 (green) on days 0, 3, 6, and 9, or left untreated, and immune responses in the colon analyzed at day 14 by flow cytometry. B, C B6 FoxP3‐GFP mice were treated and evaluated as in (A). Regulatory cytokine production in FoxP3‐GFP+ (green‐dotted bars) versus FoxP3‐GFP− (white bars) cells was measured by intracellular staining and FACS analyses (C).
The cellular colocalization of titin A168-TK with Nbr1 and p62 is MuRF1 dependent A Domain composition of Nbr1 and p62 proteins, where PB1: Phox and Bem1; ZZ: ZZ finger; NBR1: Next to BRAC1; LIR: light-chain-3-interacting region; UBA: ubiquitin-binding domain.Figure 3A Domain composition of Nbr1 and p62 proteins, where PB1: Phox and Bem1; ZZ: ZZ finger; NBR1: Next to BRAC1; LIR: light-chain-3-interacting region; UBA: ubiquitin-binding domain. The ubiquitin binding function of the UBA domain is indicated by an open triangle labeled "Ub". (B-D) Micrographs of H9C2 cells corresponding to (B) expressions of single species: MuRF1-mCherry (red), titin EGFP-A168-TK (green), Venus-Nbr1 (yellow) or Cerulean-p62 (cyan); (C) Co-expression of titin EGFP-A168-TK with mCherry-MuRF1. Neither Nbr1 nor p62 were co-transfected. The titin fragment and MuRF1 co-localize forming filamentous formations, which is in agreement with their known complexation; (D) Co-expression of MuRF1-mCherry (red), titin EGFP-A168-TK (green) and Venus-Nbr1 (yellow) or Cerulean-p62 (cyan) or respective truncated variants of the latter lacking the ubiquitin binding domain (∆UBA). (Throughout, cell nuclei are stained with DAPI; blue). (Scale bar is 10 μm in all cases).
Figure 4HeLa cells were transfected with Flag-PARP1 and Myc-BRD7 for 24 h, then treated with MG132 (10 μM) for additional 4 h, and proteins were detected by Western blot with indicated antibodies (n=3). HeLa cells were transfected with either scrambled or PARP1 siRNAs for 48 h, protein levels were detected by Western blot with the indicated antibodies (n=3). Expression of BRD7, p21 and PARP1 measured in PARP1 wild-type and knockout HeLa cells using PARP1 sgRNA by Western blot (n=3). MDA-MB-231 cell were treated with different concentration of PARG inhibitor PDD0017273 for 48 h, cell lysates were analysed by Western blot (n=3). MDA-MB-231 cell were treated with 10 μM of PDD0017273 for indicated times, cell lysates were analysed by Western blot (n=3). Wild-type and PARP1 knockout HeLa cells were incubated with 10 μg/ml cycloheximide (CHX) for the indicated times. Lysates were harvested and analysed by Western blot. Quantification of BRD7 protein are shown in F (n=3) and results represent mean ± SEM. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h of Control or sgPARP1 cells, respectively. PARP1 wild-type and knockout HeLa cells were transfected with HA-ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h, cells were lysed with RIPA buffer; followed by anti-BRD7 IP and analysed by Western blot with the indicated antibodies (n=3). SFB-BRD7 stably overexpressing HeLa cells transfected with scramble or PARP1 siRNA for 24 h were transfected with vector or HA-ubiquitin (Lys48 or Lys63 only) for 24 h, and MG132 (10 μM) was added for an additional 4 h, lysed with RIPA, subjected to IP using S tag beads followed by Western blot (n=3). HeLa cells were co-transfected either wild-type or BRD7 mutant with Flag-PARP1 for 24 h and analysed by Western blot (n=3). HeLa cells were co-transfected with either wild-type or BRD7 mutant plus HA-ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h and lysed with RIPA buffer, followed by anti-Myc agarose IP and analysed by Western blot with the indicated antibodies (n=3).