EMBO/sd-character-level-ner huggingface dataset:
Figure 6RPE cells arrested in mitosis by nocodazole (NZ) were treated with DMSO, PLK1 inhibitor BI2536 (100 nM), Aurora-A inhibitor MLN8054 (0.25 μM), CDK1 inhibitor RO3306 (20 nM) or CK1 inhibitor PF-670462 (1 μM) for 60 min, collected and analyzed by immunoblotting with indicated antibodies. Asynchronously growing RPE cells are shown for comparison (left line). Staining for H3-pS10 served as a marker of mitosis (n=3). Isolated EGFP-FAM110A-WT or -∆C proteins were incubated with active CDK1/GST-cyclin B1 in a kinase buffer containing 32P-γATP for 20 min at 30°C. Phosphorylation was detected by autoradiography and position of FAM110A by immunoblotting. Empty arrowhead indicates position of CDK1. RPE cells were transfected with control, CSNK1D or CSNK1E siRNA and nocodazole was added 12 h before collecting the cells by mitotic shake off. Whole cell lysates were probed with indicated antibodies. EGFP-FAM110A-WT, -∆Pro or -∆C proteins isolated from transfected HEK293 cells using GFP-trap were incubated with mock or with active GST-CK1δ for 20 min at 30°C. Mobility of the proteins was analyzed by immunoblotting (n=2). Cells expressing the wild-type EGFP-FAM110A or its mutants were arrested in mitosis by nocodazole and collected by shake off. Whole cell lysates were analyzed by immunoblotting. Isolated EGFP-FAM110A-WT or -S252-255A proteins were incubated with mock or with active GST-CK1δ in the presence of 32P-γATP for 30 min at 30°C. Phosphorylation of the substrate was detected by a mobility shift in immunoblotting with anti-GFP antibody or by autoradiography. Empty arrowhead indicates the same position on SDS-PAGE gel. U2OS cells were trapped in mitosis by overnight treatment with nocodazole or PLK1 inhibitor BI2536 (100 nM) and collected by mitotic shake off. Part of the nocodazole arrested cells was further treated with CK1 inhibitor PF-670462 (1 μM) for 60 min. Whole cell lysates were probed with indicated antibodies. Staining for pS10-H3 served as a marker of mitosis.
Figure 1 Figure 1. MS/MS profiling of Pb-derived MHC II immunopeptidome during blood stage malaria (A) Experimental protocol used to immunoprecipitate MHC II molecules from PbK or PbA pRBC-loaded MutuDC and to elute MHC II-bound peptides before proteomic analysis. (B) Western blot revealed with anti-I-Abβ JV2 antibody. WCL: whole cell lysate of MutuDC + pRBC ; S0: supernatant following incubation with protein-G beads ; S1: 1st wash of protein-G beads ; IP MHC II: eluates from Y-3P-immunoprecipitated MHC II, boiled or not. Representative of 2 independent experiments. (C) Venn diagram of unique peptides from P. berghei (left) and Mus musculus (right) identified in the experiments performed with PbK pRBC (blue) and PbA pRBC (orange).
Figure 1A Schematic domain organization of S. aureus Bap. S, signal peptide (aa 1-44); A, region A (aa 45-360); B, region B (aa 361-818); SP, spacer region (aa 819-947); C, repeat region (aa 948-2139); D, serine-aspartate repeats region (aa 2148-2208); W, cell wall anchor region. The aggregation-prone regions of BSP consists of 5 modules: 1 (magenta), 2 (orange), 3 (green), 4 (blue) and 5 (cyan) based on its crystal structure. B A topology diagram of BSP. C Overall structure of Ca2+-bound BSP monomer. Ca2+ ions are shown as gray spheres. Color code is the same in (A-C).
Figure 7A. Linear representation of the structure of EML4-ALK V3 and EML4-GyrB fusion proteins including residue numbering indicating domain boundaries. B. HEK293 cells transfected with YFP-EML4-GyrB and either untreated (DMSO) or treated with Coumermycin A1 to induce dimerization of GyrB before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C. Violin plot showing the number of cytoplasmic foci per HEK293 cell transfected with YFP-EML4-GyrB. Data represent counts from >30 cells, n= 4. ****P< 0.0001 in comparison to DMSO by unpaired t test. D. Schematic model showing the mechanism of EML4-GyrB foci formation. EML4-GyrB foci are formed by dynamic cross-linking interactions between the EML4 trimerization domain (TD) and dimerization of GyrB domain in the presence of coumermycin A1.
Figure 3(A) A scheme showing the FRET reaction. (B-C) Represented images of cellular interaction between TLR2 and TLR6 (A) and between F4/80 and TLR2 (B) in BM-derived macrophages with the indicated treatments were observed by Fluorescence Resonance Energy Transfer (FRET) using ImageStreamX. Scale bars 10μm. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with 500ng/ml LTA for another 0.5h at 370C. Cells were probed with anti TLR6-PE or anti F4/80-PE conjugated antibody (donor) and anti TLR2 antibody following by staining with APC-labeled secondary antibody (acceptor). PE intensity (middle panel) and FRET intensity (right panel) were measured via ImageStreanX imaging flow cytometer. The FRET intensity shown are mean ± S.E.M of two independent experiments (n=2579-15567). (D) Represented image of ERK1,2 phosphorylation levels in CD115+BM monocytes cells upon LTA exposure. Cells were pre-treated for 0.5h with 20µM of TLR2-p peptide, scrTLR2-p peptide or untreated, then washed and incubated with 500ng/ml LTA for the indicated times. ERK1,2 phosphorylation levels and total ERK1,2 levels were detected by western blotting. Equal loading was detected by measuring anti tubulin. Results are representative data of 2 independent experiments. The bands intensity was quantified for p-ERK levels after 15min of LTA exposure. Results are normalized to tubulin levels and are the mean of 2 experiments ± S.D.M.
Figure 6A-C. Fe content of whole plants (A) and the photochemical efficiencies of PSI and PSII [Y(I) and Y(II)] (B and C) in young leaves of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d. D-F. Transcript levels of FER, FRO1 and IRT1 in the roots of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.
Figure 7(A) Schematic representation of FAM134B WT, lir mutant and delta reticulon constructs. (B) U2OS cells were transfected with empty vector control (pcDNA3), FAM134B-HA WT or mutant constructs as indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against CANX, FAM134B 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed. U2OS cells were transfected with HALO-PC2, FAM134B-HA or FAM134Blir-HA constructs, treated with 100 nM BafA1 for 6h and with CST where indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against HALO, CANX, FAM134B, LC3 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed. (D) Proposed model of collagen recognition by autophagy. After synthesis, PC chains are subjected to quality control operated by the CANX/CRT chaperone system. Properly folded PC associates with HSP47 and is then secreted, whereas the misfolded fraction is sequestered by the CANX-FAM143B complex and delivered to lysosomes through ER-phagy.
Figure 1(A) Western Blot detection of ATM pS1981, SMCHD1, TRF2 and hnRNPA1 loading control in wild-type and two SMCHD1 knockout (KO1 and KO2) HeLa cells transfected with shTRF2 plasmid or EV control. (B) Representative metaphase spreads from wild-type and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid or EV control. Telomeric signals were detected with Cy3-OO-(CCCTAA)3 and are false colored in red, DNA is stained with DAPI and is false colored in cyan. Scale bar: 5µm. (C) Quantification of telomere fusions from experiment Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>3,000 telomeres counted/condition/experiment). (*) P < 0.05; unpaired two-tailed Student's t-test. (D) Representative cell cycle profiles from two biological replicates of WT and SMCHD1 KO HeLa cells transfected with shTRF2 plasmid or EV control. Cells were stained with propidium iodide and analyzed by Flow cytometry.
a, Alteration of both LC3 conversion and translocation in Ambra1gt/gt embryos.figf4a, Alteration of both LC3 conversion and translocation in Ambra1gt/gt embryos. Left panel: immunoblot analysis of LC3 in E14.5embryos. LC3-I to LC3-II conversion is reduced in mutant extracts (LC3-II/LC3-I ratio, as measured by densitometry, from left to right: 5%, 9.8%, 2.5%, 4.6%). B, body; H, head. Centre and right panels: sections from E10.5Ambra1+/+;GFP-LC3 and Ambra1gt/gt;GFP-LC3neuroepithelia were compared to evaluate GFP-LC3 subcellular distribution. Clusters of GFP-LC3 punctate structures (arrows) are indicated in wild-type sections. Images were optimized by deconvolution. Scale bar, 10 µm. a, Alteration of both LC3 conversion and translocation in Ambra1gt/gt embryos. Left panel: immunoblot analysis of LC3 in E14.5embryos. LC3-I to LC3-II conversion is reduced in mutant extracts (LC3-II/LC3-I ratio, as measured by densitometry, from left to right: 5%, 9.8%, 2.5%, 4.6%). B, body; H, head. Centre and right panels: sections from E10.5Ambra1+/+;GFP-LC3 and Ambra1gt/gt;GFP-LC3neuroepithelia were compared to evaluate GFP-LC3 subcellular distribution. Clusters of GFP-LC3 punctate structures (arrows) are indicated in wild-type sections. Images were optimized by deconvolution. Scale bar, 10 µm. c, Rapamycin-induced autophagy is impaired in Ambra1gt/gt murine embryonic fibroblasts (MEFs). Cells dissected from embryos with different genotypes (A-D) were treated with rapamycin, and the occurrence of autophagy was analysed 48 h later by the appearance of GFP-LC3 punctate staining. Representative results are shown in the upper panels, whereas the lower panel shows the data from three experiments. Scale bar, 20 µm. a, AMBRA1-BECN1 interaction by yeast two-hybrid assay. Yeast cells were co-transfected with the indicated plasmids and plated in medium with (+) or without (-) histidine (H) and adenine (A). AD, activation domain; DBD, DNA-binding domain; G4, Gal4; LAM, lamin.
Figure 5A: Superimposed structures in ribbon representation of N-CTD from SARS-CoV-2 (pink), SARS-CoV (PDB: 2CJR, cyan) (Chen et al, 2007), MERS-CoV (PDB: 6G13, orange) (Nguyen et al, 2019), and HCoV-NL63 (PDB: 5EPW, blue) (Szelazek et al, 2017). B: Electrostatic surface views of the four N-CTD structures. Red and blue colours indicate negative and positive potential, respectively.
Figure 4Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from lysates of B16-F10 cells (n=4) or B16-F10 tumors [CntlKD (n=5), BNIP3KD (n=5) and ATG5KD (n=7)]). The HIF-1α-OH /HIF-1α ratio is shown in a bar graph below. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band. Analysis was performed as indicated in the Data Information section, unless for HIF-1α/Actin ratio (in vivo), which was analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test, and LDHA/Actin ratio for shATG5 condition (in vitro), which was analyzed with Wilcoxon's rank test. Immunohistochemical staining for HIF-1α (brown) representative from CntlKD, BNIP3KD and ATG5KD, B16-F10 tumor tissues. Higher magnification of the highlighted (with a white square) stained sections is shown below. Glut1 , Pdk1 transcript levels from lysates of B16-F10 tumors [CntlKD (n=6), BNIP3KD (n=6) and ATG5KD (n=7)]. Glut1 were analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test. Vegfa transcript levels from lysates of B16-F10 tumors [CntlKD (n=6), BNIP3KD (n=6) and ATG5KD (n=7)]. Vegfa were analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test. Double immunostaining images for CD31 and αSMA quantification of corresponding tumor vessel number (F), size (G) and maturation (H) in B16-F10 tumors (n=9 per cohort). Vessel maturation (H) was analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test. Quantification of hypoxic (pimonidazole positive) area in B16-F10 tumor tissues [CntlKD (n=9), BNIP3KD (n=9) and ATG5KD (n=8)].
Fig.Figure 4(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 6 h. Cells were stained with BODIPY and visualized by fluorescence microscopy. Scale bar, 5 μm.
Figure 4Staining of bronchoalveolar lavage cytospins from WT and DKO mice with the lipophilic dye Oil Red O. Quantification of Oil Red O-positive cells in WT and DKO mice for individual animals. Results pooled from two independent experiments; 4 WT and 5 DKO mice were analyzed in total. Flow cytometric analysis of AMs from WT and DKO mice stained with the lipophilic dye BODIPY 493/503. Representative results of two independent experiments with three mice per group. Analysis as in C performed with WT:DKO mixed BM chimeras. Gating on CD45.1 and CD45.2 was applied to identify cells of WT and DKO donor origin. Representative results of two independent experiments with at least three chimeras. RNA-seq analysis of Acaa1b and Hsd17b4 expression in WT and DKO AMs, which were isolated by flow cytometry from mice in steady state (top) or from mixed BM chimeras (bottom).
Figure 1A, B Western blot analysis of ΔKu80, PKAc1-3Ty (A) parasite lysates probed with αTy antibodies. Profilin (αPRF) served as loading control. A, B Western blot analysis of ΔKu80, PKAr-3Ty (B) parasite lysates probed with αTy antibodies. Profilin (αPRF) served as loading control. C, D Peripheral localization of PKAc1-3Ty (C) shown by IFA using anti-Ty antibodies. Antibodies against GAP45 and IMC1 were used as markers of plasma membrane and IMC, respectively. C, D Peripheral localization of PKAr-3Ty (D) shown by IFA using anti-Ty antibodies. Antibodies against GAP45 and IMC1 were used as markers of plasma membrane and IMC, respectively. E Plasma membrane stained with an αSAG1 antibody is separated from the IMC stained with anti-Ty after aerolysin treatment, indicating that PKAr-3Ty localises at the IMC. F The N-terminus putative acylation site of PKAr is essential for its IMC targeting. G A second copy of DDmyc-PKAc1 is stabilised after 1 hour in presence of Shld-1.  H A second copy of DDmyc-PKAc1 stabilised with Shld-1 is targeted to the IMC in presence of a second copy of PKArWT-Ty. When of a second copy of PKArG2A-Ty is provided, stabilised DDmyc-PKAc1 does not localise at the cell periphery. Data information: Scale bars = 2 μm.
Figure 1A. Maximum likelihood phylogenetic tree generated from 61 aligned TssA sequences belonging to the indicated bacterial species. The three TssA proteins from P. aeruginosa (PaTssA1, PaTssA2 and PaTssA3) and E. coli TssA (EcTssA, GenBank accession number: 284924261) are indicated in bold. B. Graphical representation of the H1-T6SS gene cluster in P. aeruginosa (P.a) (Filloux et al., 2008). The generic tss name and the PA number of corresponding genes are indicated. Genes encoding putative baseplate components are shown in magenta with tssA1 highlighted in bold. C. Proteins from whole cell extracts and culture supernatants of PAKΔretS, the derivative tssA1 mutant (ΔA1) (both carrying the pBBR plasmid) and the complemented ΔA1 mutant carrying pBBR-tssA1 (ΔA1+A1) were analysed by Western blot. Polyclonal antibodies directed against TssA1 (A1), Hcp1, VgrG1a and Tse3 were used. The anti-VgrG1a antibody detects VgrG1a, VgrG1b and VgrG1c, as indicated on the left. Cytoplasmic RNA polymerase (RNAP) was monitored using monoclonal antibody directed against its β-subunit.
Figure 2M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured SH-SY5Y cells (A) Autofluorescent lipofuscin appears in the green channel in human brain. Scale bar, 20 µm (A) M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured R6/2 mouse cortex and striatum (B) Scale bar 10 µm M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured human HD frontal cortex (C). Autofluorescent lipofuscin appears in the green channel in human brain. Scale bar, 10 µm (D) Htt-Q25- or Htt-Q97-expressing HEK293T were analyzed by filter retardation assays. As indicated, the M1 ubiquitin antibody was preincubated with recombinant tetra-ubiquitin linked via M1 (4 x M1 ubiquitin) or K63 (4 x K63 ubiquitin).
a, RAW-GFP–LC3 cells were treated with chloroquine (50 &mgrM, 3 h) or rapamycin (200 nM, 6 h), or starved (6 h).figf2e, The percentage of phagosomes (n ≥ 50 per group) with GFP-LC3 in wild-type and Tlr2-/- macrophages. Means and ranges are shown (4 mice per group were used).
Figure 6(A) Plasma serine interpolations for each of the nine subjects. (B) Model fit to the target concentration curve (mean interpolation of plasma serine over 24 hours) with standard deviation indicated in gray. (C) Predicted plasma serine during two weeks of simulated twice-daily supplementation: 24-hour moving average value indicated in red and continuous/momentaneous indicated in gray. (D) Plasma L-carnitine interpolations for each of the nine subjects. (E) Model fit to the target concentration curve (mean interpolation of plasma L-carnitine over 24 hours) with standard deviation indicated in gray. (F) Predicted plasma L-carnitine during two weeks of simulated twice-daily supplementation: 24-hour moving average value indicated in red and continuous/momentaneous indicated in gray. (G) Plasma NAC interpolations for each of the nine subjects. (H) Model fit to the target concentration curve (mean interpolation of plasma serine over 24 hours) with standard deviation indicated in gray. (I) Predicted plasma NAC during two weeks of simulated twice-daily supplementation: 24-hour moving average value indicated in red and continuous/momentaneous indicated in gray.
Figure 7(A) Confocal image of the central retina, exhibiting endogenous ReaChR-mCitrine fluorescence (mCit) in ganglion cells, located within ~500 µm from the center of the foveal pit, a part of the humanretina where midget cell density is estimated to be ~95% of total RGC population (Dacey, 1993) (arrowhead: soma; arrow: dendritic arbor). The dashed line illustrates the beginning of the foveal slope leading towards the 'cone-only' foveal pit. (B) Confocal image of the same explant at a higher retinal eccentricity (3-4 mm from the foveal center) showing ReaChR-mCitrine fluorescence in RGCs with larger dendritic arbors. The endogenous mCitrine was not amplified. Scale bars: 10 µm. PR: Photoreceptors, GC: Ganglion cells, DAPI (blue).
The dTor membrane domain and homo-oligomerization motif are important for dTor function in the fat body A) dTor mutants and chimeras.Figure 2dTor mutants and chimeras. Sig Seq, ER targeting signal sequence. Wild-type and mutant dTormGFP proteins expressed in dTorKO fat body cells. Size of individual fat body cells (C) and LD density of individual fat body cells (D). Bars show the group mean, light grey highlights 2x SD of the control mean (animals expressing the Sec61b control ER protein), and dotted red lines show dTorKO mean. These are results of n≥3 larvae (of 3 independent crosses). One-way ANOVA, with Bonferroni's post hoc test. ** p < 0.01, *** p < 0.001 and **** p <0.0001. GFP, Phalloidin and LipidTOX detect the expression of dTormGFP chimeras (green), cell boundaries (red), and LD (red), respectively, in dTorKO fat body cells.
Figure 6A Scheme showing the experimental approach followed to measure nuclear permeability by FRAP. B-D Nuclear permeability in 30-week-old wild-type (WT) and R6/1 mice striatal MSNs, CA1 and DG neurons nuclei, respectively.(B and C) N = 4 for each condition; (D) N = 5 for each condition. An average of 25 nuclei were analysed for each sample
Figure 9(A) FTCD located in mitochondria causes mitochondria aggregation during mitosis. Mitotic cells were collected by flushing from HeLa Tet-off cells in which either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO had been induced for 24 hrs. The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis. Cells in which FTCDwt-HA-MAO was induced for 28 hrs without synchronization were used as the asynchronous control group. Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA. Panels a-f display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of seven sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). (C) A single living cell expressing FTCDwt-HA-MAO was tracked during mitosis. Mitotic cells were collected by flushing from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO, and cultured in the absence or presence of DOX. The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30-40 cells were randomly taken at the 23 hr time point. From the 23 hr to 27 hr time point, cells were tracked in the bright field every 30 mins. At the 27 hr time point, confocal images were taken of the 21-38 cells that were successfully tracked. Panels a-j display representative images. Asterisks indicate the tracked cells. Scale bar = 10 μm. (D) The results of quantification of (C). The experiment was repeated five times and each line shows the average percentage of an independent experiment. The percentages of cells that underwent mitosis are as follows: 41.5 % in the DOX(-) group and 54.7 % in the DOX(+) group. (E) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis. Mitotic cells were collected from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO and cultured in the absence or presence of DOX as in (C). Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point. Panels a-t display representative images. Scale bar = 10 μm. (F) The results of quantification of (E). Cells were classified by their cell cycle phases into the following three categories: prometaphase/metaphase, anaphase/telophase and cytokinesis. Results are shown as the mean±SD of three sets of independent experiments, with 10-14 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with the DOX(+) group and with the prometaphase/metaphase category (Bonferroni method).