EMBO/sd-character-level-ner huggingface dataset:
B Potential substrate entry routes to the active site in the context of the γ-secretase structure. Assuming larger conformational changes upon substrate binding, access of substrate to the active site appears possible from both convex and concave sides of the horseshoe structure of γ-secretase (black arrows). Several hypothetical substrate transfer routes (red arrows) from the exosites in PEN-2 and NCT to the active site are shown in bottom view. Dashed red arrows indicate paths with potential barriers imposed by cytosolic loops between TMDs 2/3 and 4/5 of PS1. Catalytic residues are shown in yellow with side chains as spheres. For clarity only the TMD domain of NCT is shown.
Immunoblots with the indicated antibodies of representative lysates from individual PDX tumors treated with vehicle control or A17pro.
HIV-1-VSV-ZsGreen at various dilutions (corresponding to multiplicities of infection (m.o.i) 0.06, 0.03 and 0.02) (F) infection is reduced in differentiated THP-1 depleted of CLIP170 determined by luciferase assays or FACS analysis, respectively.
Ubiquitination of the NB domains from SUMM2 (A), RPP4 (B) and CHS1 (C). Immunoblot of bacterial lysates form E. coil strains expressing AtUBA1-S, MBP-NB-HA, AtUBC8-S, HIS-FLAG-UBQ10 and SNIPER1-myc, or strains without one of these components. Two biological repeats were carried out with similar results.
(B, C) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti-ICP27 together with anti-STING (C). The stainings were visualized by confocal microscopy. Arrowheads, colocalizations between ICP27 and STING. Scale bar 10 μm.
(A) A scheme of the IF analysis performed to calculate the total intensity of OFD1 staining at the centriolar satellites.
(B-E) Examples of lesions observed in the lungs of infected mice at higher magnification. The two top panels depict mild (B) (scale bar: 100 μm) and mild to moderate (C) interstitium thickening accompanied by dense inflammatory infiltrates predominantly localized in the vicinity of bronchioles (yellow stars) and also present around blood vessels (green stars) (scale bar: 200 μm). (D) Alveoli filled with a proteinaceous exudate containing a few cells (green arrows) (scale bar: 50 μm). (E) Discrete degenerative lesions of the bronchiolar epithelium, such as perinuclear clear spaces (blue arrows), hyper-eosinophilic cells with condensed nuclei (orange arrow) and some intraluminal fibrinous and cell debris (black arrow) in an overall well-preserved epithelium (scale bar: 20 μm).
(B). NSF was co-localized with PTP-MEG2 in the adrenal medulla. After stimulation with 100 nM AngII, NSF and PTP-MEG2 in the adrenal medulla were visualized with immunofluorescence. White arrow stands for co-localization of the PTP-MEG2 and NSF.
(B) Co-localization analysis of CGG RNA (red traces) and SRSF1 (green traces) showing overlaps of CGG RNA and SRSF1. (a.u. = arbitrary unit)
(G , H) Snapshots from live imaging of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) embryos injected with sgRNA targeting pax2a coding sequence. The anterior part of a representative control embryo (G) is shown alongside a representative crispant (H). Crispants display a strong reduction of mKO2 fluorescence, as well as an absence of GFP signal suggesting absence of thyroid (THY) tissue. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right.
(G) The ID suffices for GFP-LC3 activation. 293 cells were transfected with the indicated chimeras and GFP-LC3A or GFP-LC3B, aggregated and mounted. Representative confocal microscopy pictures are shown. Activated GFP-LC3 appears as a collapsed mass of indiscernible vacuoles.
Growth factor-depleted Huh7.5 cells were prestimulated with 2.8 pM, 28 pM, 1400 pM IFNα or left untreated and were stimulated with 1400 pM IFNα 24 hours later. IFNα-induced signaling was measured by time-resolved quantitative immunoblotting and detected with chemiluminescence using a CCD-based camera. Data was normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration.   Model calibration with time-resolved IFNα-induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively.
(F and G) (F) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr, incubated for 4 hr at 37°C or 18°C, and labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes. Histogram in (G) shows the amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment (Manders' coefficient). Error bar, SEM. NS, not significant; ∗∗p < 0.01.
(E) Mitochondrial fractions isolated from the heads of Lon-R2 and PINK1-Myc expressing flies were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (F) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel E were performed as described in B. All experiments described were repeated at least three times. Error bars represent s.e.m.
(B) Fluorescence images of cells described above. Large GFP-LC3 aggregates are indicated by arrow heads.
Representative 3D microglia reconstructions from 5 and 15 months old mice showing microglia changes 2 and 10 days post LPS injection. Scale bar: 20µm
(G) ATAC peaks of HSCs in the early or late phase, and H3K27ac peaks in CD48- HSCs or CD48+ HPCs in the late phase within the promoter and enhancer regions in the CD48 locus.
(A) Model predictions of the number of regulation steps required for permanent activation of each network component. Light blue regions indicate where only some concrete networks predict that the given gene has permanently activated
TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (A) (Left panel) confocal microscopy images of the fluorescence in U2OS cells stably over-expressing Flag-tagged-TIGAR (clone TIGAR#7) or control cells (clone Cont#1) and infected with an adenovirus expressing GFP-LC3 for 16 h. Cells were then left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.
(C) Quantification of fibrillar collagen I (Yellow-Orange) and III (Green) based on polarized Sirius Red images from B. Each dot represents a mouse (n=11 per group).
A. Representative western blot showing the changes of O-GlcNAcylation in primary human renal proximal tubule epithelial (HRPTEpi) cells (originating from fetal or adult kidney) treated with different concentrations (0-10 ng/mL) of Stx2a for 24 h.
C The interaction between PKR and lincRNA-EPS were checked by RIP-qPCR using anti-PKR antibody, and the efficiency of IP was verified by Western blot.
(b) Levels of spermidine in the heads of wild-type flies fed with food supplemented by 1 or 5 mM spermidine (Spd1mM+ or Spd5mM+, respectively) or food with no spermidine (Spd−). Data are shown normalized to spermidine levels of 1-d-old (1 d) Spd− flies (n = 4 independent experiments; F = 14.08 for Spd− flies, F = 5.30 for Spd1mM+ flies, F = 2.62 for Spd5mM+ flies; one-way-ANOVA with Bonferroni correction). 15 d, 15 d old; 30 d, 30 d old.
GO and KEGG enrichment analysis of differentially expressed genes based on RNA-seq data with adjusted-P value.
A. Reconstitution of Smc5/6 and Nse5/6 complexes. Left panel, schematic depiction of the composition and organization of the yeast Smc5/6 'core' hexamer and the Nse5/6 dimer. Middle panel, elution profiles for analytical gel filtration (Superose 6 Increase 3.2/300) of the Smc5/6 core hexamer, the Nse5/6 dimer and a holo-complex obtained by mixing of dimer and hexamer. Measured by absorption at 280 nm. Right panel, peak fractions were analysed by SDS-Page and Coomassie Brilliant Blue staining.
HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). E Cell survival was evaluated by CellTiter-Glo after 24 hours of treatment (means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA).
E, F Topology diagrams of the β-grasp folds of ubiquitin (E, left), the ubiquitin-fold domain (UFD) of the E1 enzyme (E, right), and the putative Ubl domain in PknG (F, left) are shown, and a cartoon depiction of the β-grasp fold of PknG is included (F, right). Strands in the topology diagrams are illustrated as cyan arrows, and the helices are highlighted in magenta.
(A) Immunodetection of p‐PERK and p‐eIF2α in Mfn2 KO MEFs, Mfn2 knockdown 3T3‐L1 fibroblasts, and Mfn2 knockdown C2C12 myoblasts.
Heatmap of key pluripotency and differentiation associated gene expression during EB formation from WT and hnRNPLL-KO clones (H8 and G6).
D Flow cytometry analysis of neutrophils in hearts and bone marrow under inverted light cycle conditions. Two-way ANOVA followed by Bonferroni post-hoc test; n = 3 mice for no MI in both ZT groups, n = 5 for ZT5 and n = 6 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0147 (24h MI).
(D, E) Infiltration of F4/80+ macrophages determined by flow cytometry. (WT, N = 10, TNCKO, N = 7 tumors), * p = 0.0304, unpaired t-test and iNOS+ macrophages (WT, N = 9, TNCKO, N = 7 tumors) * p = 0.0197, unpaired t-test. Mean ± SEM.
B Corresponding queuosinylation and methylation levels, as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).
(c) Model of the position of TREM2-TMH wt and K186A within the membrane. The amino acids in the kink region are exposed to the solvent in the wild-type case but buried in the membrane for the K186A variant.
The maximal biomass concentration observed for M. florum cultures growing in CSY medium with varying initial concentrations of sucrose. Biomass was measured using colony forming units (CFU/ml; left axis) and converted to grams of dry weight (gDW/L; right axis). A rectangular hyperbola fit is shown (yellow), and the dotted line represents the maximal biomass value predicted by the fit (1e8 x 5.95 CFU/ml; 0.013 gDW/L).
(B) Fluorescent microscopy images of representative embryos expressing each GFP reporter and the dsRed control 24 hours post injection. uORFs and oORFs repress downstream translation as predicted by analysis of endogeneous transcripts. Repression is observed in reporters with uORF/oORF-containing TLSs, but the effect is weaker for a uORF with a bad initiation context. Group pictures can be found in supplementary figure S2.
(E) The localization of p150glued in centrinone-treated acentrosomal spindles of HeLa cells. Red, gray, green and blue represent NuMA, p150glued, Cep152 and DNA, respectively. Scale bar, 5 μm.
(A) A schematic of the tandem ORF assay. The Rluc ORF (orange) and Fluc ORF (light green) are separated by two P2A translation skipping sequences (light blue). A sequence to be tested (shown as X in gray) is inserted between the two P2A sequences.
H, I. Double staining for insulin (green) and Pbk (red) of mice islets after 12 weeks of vehicle (H) or MI (I) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). Pbk staining is indicated by arrows. Scale bar: 50 μm. J. Quantification of Pbk staining-positive β-cells. Five mice for each group, and at least 10 islet images per mouse were analyzed. *P = 0.0120 (two-tailed unpaired student's t-test).
A Yeast two hybrid (Y2H) assays of the extracellular juxtamembrane domains of HERK1 and ANJ (HERK1exJM and ANJexJM, respectively) with LRE (residues 23-138; signal peptide and C-terminal domains excluded).
B The number of PDGFRα+ cells in four microscopic fields was assessed for co-staining with an anti-Ki67 antibody to reveal proliferating cells. P values were calculated by unpaired Student's t‑test (n=4 fields for each group, trends are representative of those observed in two other experiments).
(H) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells pre-infected with ZIKV (MOI=1) or mock control for 24 hrs. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) stimulation for 6 hrs.
F Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates as in (E). No statistical difference (P<0.05 cutoff) was detected by ANOVA. N=4 trials.
(A, B) Immunoblot analysis of the phosphorylation level of CLIP-170, AMPK and ACC in heart (A) and liver (B) along with the developmental stages. α-tubulin was used as a loading control. (C-E) Quantitative analysis of western blotting shown in Fig 1A. (F-H) Quantitative analysis of western blotting shown in Fig 1B.
Lysosomes, isolated as in (B), were incubated with the CMA substrate GAPDH, and CMA activity was evaluated as in Fig8 and as described in Materials and Methods.Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors. Results are mean ± SEM (n = 4). *P = 0.0126 for WT versus Ctns−/− and *P = 0.0436 for WT versus Ctns−/− + cysteamine; NS, not significant; unpaired t-test.
E. Differential GSEA was performed on combined genotype comparison [all]/Act vs [all]/wt and two datasets of human AK vs normal skin (GSE2503; Nindl et al, 2006) and GSE63107 to compare these transcriptomes to gene sets derived from top upregulated genes in these datasets (top); and gene sets derived from top upregulated genes in datasets of differential human macrophage activation (IFN-γ, TNF-α, LPS, IL-4 vs non-treated) and selected human myeloid/lymphoid cell type-specific gene expression profiles (macrophage vs monocyte, dendritic cell, and T-cell) (Xue et al, 2014; bottom). Activin target genes enriched in the AK transcriptomes are shown (right). Data are presented as pseudo-heatmaps with magnitudes of Normalized Enrichment Scores (NES) and Log2(Fold Change) color-coded as indicated in the respective legends; all non-grey comparisons are FDR<0.05 for NES and p<0.10 for Fold Change.
A: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4-, 12-, 18- and 24-month-old mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. B: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4- and 12-month-old irradiated (RT+) and non-irradiated (RT-) mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test.
D An inhibition-zone assay showing the inability of AMSIN to bind DNA. The bacterium used was MRSA P1374 and the peptide dose was 1.0 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann-Whitney U (ns: no significance
(A) Following incubation for 1.5 h with buffer in the absence or presence of 10 µM hGBP1F and 5 mM GTP, live bacteria were treated with antibiotics (2.5 µg/ml PMB or 5 mg/ml lysozyme) for 30 min or left untreated. The number of viable bacteria was subsequently determined by colony forming unit (CFU) counts. Graphs show mean CFUs ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ****, P ≤ 0.0001.
CUT1 and CUT2 domains are required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from HeLa cells transfected with V5-tagged Satb2346-733 and Satb2616-733 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells expressing the CUT1 and CUT2 containing deletion mutant (Satb2346-733).
(A) Two sets of FTCD molecules and a p97/p47 complex form a quaternary complex. His-tagged FTCD was biotinylated and immobilized on streptavidin beads (His-FTCD-beads). The beads (1.0 μg) were incubated with Flag-tagged FTCD (2.0 μg) in the absence or presence of p47 (1.0 μg) and p97 (2.0 μg). The blots were probed with HRP-conjugated avidin and antibodies to Flag-tag, p97 and p47.
E. Time course analyses of MAM-specific proteins at MAM and in the whole lysates of SOD1G93Amousespinal cords.
E. Relative invasion of wt- or T693A-transfected BT20 cells into fibronectin-supplemented collagen I was quantified as described in Material and Methods. Graph depicts mean ± S.E.M. of N=8. *P value=<0.05 (Student's t-test).
(I) Schematic model for the shuttling of LSD1 from parent cells to recipient cells using sEVs as vehicles. The sEV-delivered LSD1 promotes recipient gastric cancer stemness and chemoresistance.
(A-J) Confocal z-projections of brains expressing MiMIC mediated Dpr12GFSTF (Dpr12-GFP) and DIP-δGFSTF (DIP-δ-GFP) fusion proteins of the indicated genotypes and time points. (A-D) Dpr12-GFP expression is diffuse in DIP-δT2A-Gal4 homozygotes mutant brains at L3 (A; n=20/20), 24hr APF (B; n=14/14), 48hr APF (C; n=28/28) and adult (D; n=26/26). (E-H) DIP-δ-GFP expression in dpr12∆50-81 homozygotes mutant brains remains localized to the distal part of the γ-lobe at L3 (E; n=16/16), 24hr APF (F; n=16/16), and 48hr APF (G; n=10/10) but cannot be identified in adult brains (H; n=16/16). (I-J) Dpr12-GFP expression in WT animals (I, n=8/8) or in those ectopically expressing DIP-δ in PAM-DANs that innervate the γ3 zone (J, n=14/14) driven by MB441B-Gal4 (PAM-DAN-γ3-Gal4). DIP-δ expression in PAM-DAN-γ3 resulted in Dpr12-GFP localization within the γ3 zone (arrow), in addition to its normal γ4/γ5 localization.
C) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins. β-actin was used as an internal loading control. Mitochondrial fraction (bottom panel) isolated from HepAD38 cells (see the purity in Figure 2B) was analyzed by Western blotting with phospho-Drp1 (Ser616) antibody.
A, B, C. Cell lysates of HEK293 cells co-transfected with Myc-ATG13 (C) and either empty vector control, FLAG-C9orf72S or FLAG-C9orf72L were subjected to immunoprecipitation with anti-FLAG (C) antibodies. Immune pellets were probed for FLAG-C9orf72 (C), Myc-ATG13 (C) on immunoblots.
Decreased Drp1S616 phosphorylation in PINK1-null HEK293 cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) HEK293 cells (a) and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Two independent PINK1KO and parkinKO HEK293 cell lines (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.
D H2A.B interacts with Pol I. Western blot showing co-immunoprecipitation of RPA194 with H2A.B. The Pol I subunit RPA194 was immunoprecipitated from L1236 (left panel) and L428 (right panel) nuclear extracts. The pull-down by RPA194 antibody (IP), pull-down by empty beads or IgG isotype control, and 1% input protein were loaded for Western blot detection using anti-H2A.B and anti-RPA194 antibodies.
A-F (A, D): Average all-atom atomic fluctuations (RMSF; root mean square fluctuations) for SecY (A) and SecE (D). The mean RMSFs are projected onto the 3D structures of SecY/SecE and colored according to the color scales starting from blue (low mobility) to red (highly mobile). (B, E): Overlay of the SecY (B) or SecE (E) representative structure (red) onto the corresponding starting structure (dark cyan). Parts that show a pronounced structural change are explicitly shown as cartoon representation, and the movements are highlighted by arrows. (C, F): Visualization of displacements along the 1st (golden arrows) PC computed for the joint, five 1 µs long MD simulations for SecY (C) and SecE (F). The amplitudes of the motions were scaled, and a cutoff for small displacements was applied for best graphical representation. Distribution of lipids during MD simulations. Grids represent the 3D density of phosphatidylcholine (PC; blue) and phosphatidylglycerol (PG; red) groups from the lipid bilayer. The densities were normalized to the number of considered conformations, which are identical in both cases.
(A) EM scan of a wt (left) and ftfd mutant ommatidium (right) of a ftfd clone after 14 days. Scale bar: 1 μm. High‐magnification panels (far right, top to bottom) display autophagosomes with undigested debris, damaged mitochondria and forming phagophores (arrowheads), found in ftfd mutant cells. Scale bar: 0.2 μm for zoom‐in panels.
(C) Vti1b trafficking was monitored by FRAP analysis in WT (untreated and cholesterol treated) and MSD (untreated and MβCD treated) MEFs transfected with GFP-Vti1b (see Materials and methods section for details). FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach (100%) and are representative of 10 recordings from different cells.
(G-H) Scramble and PGC-1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses (G) and quantification of the indicated proteins (H)
Single-cell pS6(Ser235/236) levels in the c-JunHigh and c-JunLow fractions of cells (as measured by single-cell multiplex immunofluorescence microscopy) after 48 h of JUN knockdown and 24-h treatment with 0.32 μM vemurafenib. Single-cell pS6 data are presented as box-and-whisker plots with median signal intensities and interquartile ranges; bars extending to 1.5× the interquartile range are shown for each condition as a measure of variance. P-values were calculated using a two-sided nonparametric Mann-Whitney U-test.
Immunofluorescence analysis of GBP1-5, was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).
A. WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes.
A Heatmap showing codon occupancy according to the color scale. Green bars indicate Q-decoded codons. The Asp-GAC codon translated by Q- and m5C38-tRNAAspGUC is shown in yellow and Lys and Glu codons are in orange. Lack of Q reduces ribosome translation speed at all Q dependent codons and at near-cognate decoded codons. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).
Lysates from Hela cells co-transfected with LAMTOR1-Flag or its mutants ∆N and ∆C and TRPML1-YFP were immunoprecipitated with anti-GFP or control IgG antibodies and probed with the indicated antibodies. Left, input proteins; right, immunoprecipitated (IP) proteins.