EMBO/sd-character-level-ner huggingface dataset:
The IgSF domain in the β protein of GBS binds to the N-terminal domain of CEACAMs.Figure 2Schematic representation of the β protein (Hedén et al, 1991). The first residue of each domain is indicated. XPZ is a proline-rich repeat region (Areschoug et al, 2002a). Known human ligand binding partners are indicated above each respective domain. IgA binds to a region denoted IgABR. Factor H binds the β75K region, but does not bind via XPZ. Staining of recombinant β protein domains (B6N, IgABR, B6C, β-IgSF and β75KN) after separation by SDS-PAGE. Recombinant proteins were expressed in E. coli. β protein purified from GBS is used as a control. Dotted boxes signal proteins of expected size. Binding of rCC1-His or rSiglec5-Fc to dynabeads (DB) coated with β protein domains. Mean and SD values are reported for n = 2 replicates. Binding of varying concentrations of β-IgSF or human serum albumin (HSA) coupled to streptavidin-PE (SPE), to DB coated with CC1 (DB.CC1) or control protein (DB). Mean and SD values are reported for n = 6 replicates. Structure of the relevant (Siglec-like sialic acid-binding) domain of the highest-scoring template 5KIQ (SrpA binding region), and predicted three-dimensional model of the IgSF domain from the β protein, constructed from the Hhpred alignment using MODELLER.(Webb & Sali, 2016) Inhibition of β-IgSF binding to DB.CC1 by anti-CC1-N mAb (clone CC1/3/5-Sab), but not by anti-CC1-A1B1 mAb (clone B3-17). β-IgSF-biotin was coupled to SPE. Mean and SD values are reported for n = 6 replicates. Concentration-dependent binding of β-IgSF to DB coated with 100 μg/ml CC1 (DB.CC1), CC1-N (DB.CC1-N) or CC1-A1B1A2 (DB.CC1-A1B1A2). Assays utilized proteins coupled to SPE. Mean and SD values are reported for n = 6 replicates.
Figure 5(A) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with NCoR1-Flag and/or MEF2a-HA or with empty plasmids. Student's t test was used for statistical analysis (B) Co-immunoprecipitation (Co-IP) analysis of NCoR1 and MEF2a in HEK293FT cells transfected with full-length NCoR1-Flag and MEF2a-HA. (C) Co-IP analysis of NCoR1 and truncated MEF2a in HEK293FT cells. Schematic illustration of MEF2a-HA construct is shown above the Co-IP results. (D) Co-IP analysis of MEF2a and truncated NCoR1 in HEK293FT cells. Schematic illustration of NCoR1-Flag construct is shown above the Co-IP results. (E) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with full-length MEF2a and domain-specific NCoR1. Student's t test was used for statistical analysis (F) Western blotting analysis of NCoR1-flag in NRVMs infected by control lentivirus (Control) or NCoR1 (1939-2453)-flag lentivirus [NCoR1(1939-2453)-OV] for 4 days. (G) Representative immunofluorescence staining of α-Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle (DMSO) or PE for another 48h. Scale bar: 50μm. (H) Quantification of the surface area of α-Actinin-positive NRVMs. A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis (I) qRT-PCR analysis of Acta1 and Nppa in NRVMs. n=4:4. Data represent three independent experiments. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis
a, LAMP1/LC3 NRK cells were starved for 4 h and treated with GTP&ggrS for 6 h of further starvation.figf4a, LAMP1/LC3NRK cells were starved for 4 h and treated with GTPγS for 6 h of further starvation. b, Rab7Q67L-GFP-transfected LC3-CFP/Lamp1-RFP NRK cells starved for 10 h. c, Rab7-CFP NRK cells starved for 2 h and treated with 100 nM rapamycin for 8 h of further starvation. d, LAMP1-YFP NRK-LC3 cells starved for times shown with 1 µg ml-1 leupeptin and blotted with indicated antibodies. e, Cells in d imaged.. f, Fibroblast cells from patients with Scheie syndrome (GM01256) or healthy controls starved and blotted with indicated antibodies. g, Cells in f imaged. Lysosome size classified by Image-ProPlus (IPP) software. h, Provisional model of the autophagic lysosome reformation (ALR) cycle. Scale bars, 5 µm.
Figure 2(A and B) Binding affinities in Kd are shown for baricitinib (A and B), tofacitinib, upadacitinib, AZD7762 (positive control), and broad-spectrum tyrosine kinase inhibitors sunitinib and erlotinib (B). Kd values were estimated from an 11-point concentration response curve with fixed top parameters and a four-parameter logistic model. Kd, equilibrium affinity constant (n=3) (C) Protein and mRNA expression of relevant NAKs in primary human liver organoids (n=3) (p<0.05). The immunofluorescence shows the juxtaposition of 1A9 (signal from the SARS-CoV-2) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain (n=1). Colocalization of 1A9 and ACE2 is depicted with an arrow. (D, E) Time course of days (one, three, and six days) post infection (dpi) using liver spheroids and SARS-CoV-2 at 0.1 and 1.0 multiplicity of infection (MOI) (n=1). (F) Viability of spheroids was quantified by measuring intracellular ATP concentrations. Note that no significant (n.s.; Student's t-test) decrease in cell viability was observed for any concentration tested (n=10). (G) Schematic showing the timeline for the experiment shown in panel H. (H) Viral load in infected (MOI 0.1) spheroids as measured by qPCR after treatment with 400 nM and 800 nM of baricitinib 48 hours after infection (*p<0.05; Student's t-test). Data are mean ± standard error of mean (n=3).
Figure 4A Immunoblot analysis of TRAF6-mediated ubiquitination of wild type, and mutant FOXP3 molecules. 293T cell lines were transfected with a normal HA-tagged Foxp3 construct, another encoding a ubiquitination resistant mutant in which all lysine residues were replaced by arginines (20R), or one of 20 single lysine-containing constructs with only the indicated lysine residue available for modification. These cell lines also received expression vectors encoding TRAF6 and ubiquitin molecules labeled with Myc and FLAG tags, respectively. Negative controls did not receive TRAF6 or expressed FOXP3 alone. Labeled FOXP3 proteins were pulled down from cell lysates (anti-HA) and ubiquitinated species were visualized by immunoblotting for FLAG. B Assessing the impact of K262 mutation on FOXP3 ubiquitination by TRAF6. 293T cells were transfected with combinations of expression constructs encoding Myc-TRAF6, FLAG-ubiquitin, and an HA-tagged FOXP3 molecule that was either normal (HA-FOXP3) or possessed a K-to-R mutation at residue 262 (K262R). FOXP3 proteins were pulled down with anti-HA beads and either ubiquitinated proteins or total FOXP3 proteins were detected by probing for FLAG and HA, respectively.
A-bromodomain is required for PCa stem cells A.Figure 4A. RNA-seq showing differential gene expression between PC3-CSCs and the parental PC3 cells (P<0.05, Log2 fold change 1). B. Top ten biological process gene ontology (GO) terms as determined using DAVID 6.8 for genes downregulated and upregulated in PC3-CSCs relative to parental PC3 cells. C. qRT-PCR of genes differentially expressed in PC3-CSCs compared to PC3 cells showing the upregulation of CSC markers (ALDH1A2, LGR5, CD24) and downregulation of genes related to developmental processes (HOXA1, HOXA4, SOX7). Data are from two independent experiments. mRNA levels were normalized to GAPDH. D. BAZ2A is required for the dedifferentiation of PC3 into PC3-CSCs. Left panel: Representative images from two independent experiments showing the impairment of PC3 cells to form tumorspheres upon stable depletion of BAZ2A. Right panel: BAZ2A mRNA levels in three independent PC3 cell lines stably expressing shRNA-BAZ2A were measured by qRT-PCR and normalized to GAPDH mRNA from 2 independent experiments. E. Left panel: qRT-PCR showing BAZ2A mRNA levels in PC3 cells depleted of endogenous BAZ2A with siRNA specifically targeting human BAZ2A sequences and expressing mouse BAZ2Awt and BAZ2A-BRD mutant (BAZ2AY1775F). Values were normalized to MRPS7 mRNA. Data are from 3 independent experiments. Right panel: Quantification of tumorspheres from 3 independent experiments. Statistical significance (P-values) was calculated using two-tailed t-test (* <0.05; **< 0.01). Error bars represent SD.
a inhibits CSR.Figure 1(A) Representative FACS profiles of IgA switching in CH12F3-2A cells. The control and Phf5a siRNAs are indicated at the top. The number in each FACS profile indicates the percent (%) IgA+ cells at 24 h and 48 h after CIT stimulation. (B) Top: Illustration of Phf5a with a central PHD domain. Bottom: Confirmation of Phf5a KD by Western blot. (C) CSR complementation assay using siRNA-resistant Phf5a with Myc and Flag (MF) epitopes tagged at the C-terminus. (D) Schematic of MF-tagged siRNA-resistant Phf5a (Phf5aR-MF). Asterisks (*) indicate the approximate locations of mutations in the cDNA made to generate the siRNA-resistant version. Western blot confirmed the KD efficiency of the endogenous Phf5a, and the expression of Phf5aR-MF. (E) Quantitative RT-PCR (qRT-PCR) of μGLT, αGLT, and AID from siControl- and siPhf5a-treated samples. The values are presented as mean ± sd (n=3). (F) Ethidium Bromide-stained gels showing the PCR detection of excision circular DNA (αCD) and Gapdh as a control in the genomic DNA samples indicated.
SPPL2c cleaves SNARE proteins in testis and is involved in acrosome formation.Figure 6A) Western Blot analysis of SPPL2c in membrane preparations from HEK293 inducibly overexpressing SPPL2c, human testis and mouse testis tissue. For human and mouse tissue, 5 μg protein of each total testis preparation is loaded. The membrane fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 cells is diluted 1:50 (0.1 μg total protein). B) Western Blot analysis of selected glycoproteins in mouse tissues from two wildtype C57BL/6J (Black6) mice. Endogenous Nicastrin (NCT) and SPPL2a were analysed in membrane preparations from testis, kidney, brain, spleen and liver in littermate male mice. Note that immature proteins (im) accumulate in testis compared to other tissues. SPPL2c protein is only detected in testis tissue. C) Analysis of Syntaxin 8 (Stx8) in SPPL2c-/- mice compared to wildtype (wt) littermates. Membrane isolates from whole testis were analyzed for Syntaxin 8 (Stx8) and SPPL2c expression. TMEM192 serves as loading control. D) Syntaxin 8 protein levels were quantified from the respective Western blots by densitometric analysis and normalized to those of TMEM192. Bars represent mean values of n=3 animals ± SD normalized to mean Syntaxin 8 levels of wildtype mice. Statistical significance was calculated applying an unpaired, two-sided Student's t-test. * p≤0.05. Note that Syntaxin 8 is accumulating in SPPL2c-/- mice. E) Real-time qPCR analysis of Syntaxin 8 (Stx8) mRNA levels in testis of SPPL2c-/- and the wildtype mice normalized to that of Tuba1a. The diagram depicts mean values of Stx8 mRNA levels normalized to that of wildtype animals of n=6 individuals from two independent experiments ± SD. For statistical analysis an unpaired, two-sided Student's t-test was performed. ns = not significant. F) Immunohistochemical stainings of cross-sectioned seminiferous tubules of SPPL2c+/+ and SPPL2c-/- mice using anti-Cab45 antibody. Stages VII/VIII are depicted. Higher magnifications are indicated with dashed lines and black arrows indicate pre-acrosomal structures. Scale bar 50 µm. G) Volcano plot depicting the binding affinities of lectins on sperm lysates from SPPL2c-/- mice compared to SPPL2c+/+ (wt). Each circle represents one lectin. The lectins above the dashed line (light blue) have a significant p-value<0.05. Lectins on the left side of the blot demonstrate reduced binding while UEA-I on the right side has increased binding. n=4 animals per genotype, two-sided, unpaired Student's t-Test.
Figure 2a) Total number of significantly up- or down-regulated proteins (|z-score| >1.96, q-value <0.05) per time point of SARS-CoV-2 infected Caco-2 cells as described in 1A. Proteins that were significantly changed in abundance (left) and stability (right) are sub-grouped according to either down-regulation/destabilization (red) or up-regulation/stabilisation (turquoise). b) Gene Ontology (GO) biological process and pathway enrichment of selected pathways. See Dataset EV2 for complete GO term biological process and pathway enrichments.
Figure 6(A) Relative qRT-PCR analysis of IFNB, IFIT2 and IFIT1 mRNA levels in wild type (WT) or caspase-1 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points. (B) Relative qRT-PCR analysis of IFNB mRNA levels in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points. (C) Relative qRT-PCR analysis of IFNB mRNA levels in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points. (D) Relative qRT-PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag-NS1-inducible THP-1 cells treated with or without Dox then left untreated or treated with YVAD (20 μM) for 3 hrs, followed by ZIKV infection (MOI=1) for the indicated time points. (E) Relative qRT-PCR analysis of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs. (F) Relative qRT-PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs. (G) ELISA of IFN-β in the supernatants of PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs. (H) ELISA of IL-1β production in brain of WT or Nlrp3-/- 1-day-old neonatal mice (n=3 per group) intracerebrally injected with 1×105 plaque-forming units (PFU) of ZIKV for 5 days. (I) Relative qRT-PCR analysis of IFNB mRNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection (J) ELISA of IFN-β production in brains of WT or Nlrp3-/- neonatal mice (K) Relative qRT-PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection (L) Plaque titration of ZIKV in brain tissues of WT or Nlrp3-/- neonatal mice (M) Body weight gain of WT and Nlrp3-/- neonatal mice (n=4 per group) inoculated with ZIKV (1×105 PFUs).
Figure 5(A) GSH-Sepharose beads-immobilized full length or truncated GST-TAK1 proteins were incubated with K63-linked polyUb chains in 300 μl binding buffer, followed by washing and resolving in SDS-PAGE, and then immunoblotted with anti-ub. GST-fused NEMO serve as positive control and NEMO L329P mutant as negative control. The blotted membrane was stained with Ponceau S to demonstrate the amount of the input GST-fused proteins (lower panel). (B) Strategy for in vitro polyUb transfer experiments. (C and D) Immobilized GST-TAB2 was pre-incubated with K63-linked polyUb chains and then eluted from beads, followed by incubation with eluted GST-TAK1 and GST-p38IP as shown in (B). Incubation mixture were immunoprecipitated with anti-TAB2 or anti-TAK1, and then immunoblotted with anti-ub, anti-TAB2, anti-TAK1 and anti-p38IP. (E) Statistical analysis of polyUb binding to TAB2 or TAK1 in (C and D). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.). (F) Immobilized GST-TAB2 and GST-TAK1 were respectively pre-incubated with K63-linked polyUb chains or not as indicated, then eluted from beads, followed by coincubation as indicated. Incubation mixture were immunoprecipitated with anti-TAB2, and then immunoblotted with anti-TAB2 and anti-TAK1. The relative intensity of immunoprecipitated TAK1 was calculated and shown in the right. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.). (G) Immobilized GST-TAK1 was pre-incubated with K63-linked polyUb chains or not, then eluted from beads, followed by incubation with eluted GST-p38IP. Incubation mixture were immunoprecipitated with anti-TAK1, and then immunoblotted with anti-TAK1 and anti-p38IP. The relative intensity of immunoprecipitated p38IP was calculated and shown in the right. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).
Figure 5(A) In vivo morphology. Cells expressing the indicated SNARE variants were grown in SC medium (16 h, 30°C), stained with FM4-64 and their relative morphology was assessed by fluorescence and DIC microscopy. Scale bar: 5 µm. The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments. (B) and (C) SNARE activation on isolated vacuoles. Vacuoles were isolated from a strain co-expressing NYV1-S9-EGFP and VAM3-S9-mCitrine and from an isogenic wild-type. 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP-regenerating system and recombinant, purified Sec18/NSF (rSec18, 50 µg/ml). After 10 min of incubation at 27°C, the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1. Co-immunoprecipitated proteins analyzed by SDS-PAGE and Western blotting. The histograms provide quantifications of the band intensities as the means and s.d. from three independent experiments.
Figure 2Flow cytometric analysis of γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). In graphs, each symbol represents a mouse and line the median. Numbers of γδT17 cells in the LN and skin (staining as in Fig EV1) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 11; 4 experiments. Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm). Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 6; 3 experiments, IMQ: n = 8; 3 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.
Overview of frequently mutated genes across 3,217 SCAN-B samplesWaterfall plot of the 20 most frequently mutated genes (rows) across 3,217 SCAN-B samples (columns).Figure 3Waterfall plot of the 20 most frequently mutated genes (rows) across 3,217 SCAN-B samples (columns). Genes are ranked from top to bottom by mutation frequency. Samples are sorted by histological subtype and alteration occurrence. Mutations are colored by predicted functional impact.
Current understanding of flagellar assembly based on the canonical systems of E.Figure 1
Fig.Figure 2(A) Silver-stained PAGE gel of proteins from a nuclear extract that interact with either GST-VgpA or GST glutathione agarose. The protein bound by GST-VgpA (red arrow), but not GST, was identified as EBP2 by mass spectrometry; GST-VgpA is labeled with a blue arrow. (B) Western blot using anti-EBP2 as antibody confirmed that EBP2 binds VgpA. (C) Interaction of VgpA or VgpA mutants (VgpAΔ10 and VgpAL10A) with EBP2. (D) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes
Bax activation does not per se depend on mitochondrial Bax levels.Figure 7A Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing wild-type Bcl-xL in the presence or the absence of Bak or Bax analyzed by Western blot. Carbonate extractable supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the IMS during carbonate extraction and VDAC remains in the pellet. n = 4.B Western blot analysis of Bcl-xLTBax carbonate extraction from the heavy membrane fraction from Bax/Bak DKO cells expressing the Bcl-xL variant with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.C Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing Bcl-xL G138A with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4. D HM protein fraction and carbonate extraction of Bax/Bak DKO cells expressing Bax, BaxTBak or Bax S184V. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4. E Smac release from the heavy membrane fraction (HM) into the cytosol (C) in HCT116 Bax/Bak DKO cells overexpressing Bax and BaxTBak without apoptotic stimuli analyzed by Western blot. Bax and BaxTBak were expressed differentially to produce similar levels of mitochondrial Bax. GAPDH and Tom20 serve as fractionation controls. n = 3. F PARP cleavage in HCT116 Bax/Bak DKO cells expressing similar levels of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli. Actin is used as loading control. n = 3. G Bax (blue, left) and Bak (green, right) are retrotranslocated similarly by pro-survival Bcl-2 proteins. The shuttling shifts also OMM-integral Bax and Bak into the cytosol dependent on the interactions between hydrophobic Bcl-xL groove and BH3 motif of either Bax or Bak. While Mcl1 shuttles Bax and Bak like Bcl-xL, Bcl-2 only accelerates Bax retrotranslocation. Fast shuttling from the mitochondria into the cytosol at the level of Bax retrotranslocation in proliferating cells shifts Bax and Bak into the cytosol and inhibits their pro-apoptotic activities in parallel (top). Wild-type Bak is usually shuttled at low retrotranslocation rates and thus accumulates on the mitochondria (bottom, right). Shuttling at similar speed also causes mitochondrialBax accumulation but results in stimulus-independent Bax activation (bottom, left). Therefore cells are required to shuttle Bax at high rates to prevent commitment to apoptosis.
Hops−/− mice resistance to apoptosis.Figure 2A Reduced caspase-3 activation in Hops−/− mice upon apoptotic stimulus. Hops+/+ and Hops−/− mice were treated with etoposide for the indicated times and thymus, spleen, testis and liver were harvested and Tissue-Tek® O.C.T. embedded. Sections were immunostained with anti-cleaved caspase-3 antibody followed by appropriate secondary antibody labelling (green). The graphs display the relative positive cells number. Scale bars, 10µm. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In A data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test. B HOPS affects p53 increase after stress in vivo. Whole-cell homogenates from organs, were immediately prepared and immunoblotted with antibodies to detect p53 and GAPDH. The p53 levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 1. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In B data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.