EMBO/sd-character-level-ner huggingface dataset:
in the establishment of HIV latency.Figure 7PEBP1 interacts and inhibits the activity of Raf1 and IKK kinases. This blocks the phosphorylation of downstream proteins ERK1, IKK and IκBα, resulting in the sequestration of p65/p50 heterodimer in the cytoplasm, thereby silencing HIV-1 transcription. When PEBP1 is knocked out, Raf1 and IKK signaling pathways are activated to phosphorylate IκBα, thereby releasing p65/p50 heterodimer to translocate into the nucleus to drive HIV-1 transcription.
Figure 2A. Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non-reducing conditions before or after removal of N-glycans by PNGaseF. Blots were decorated with anti-µ antibodies. Blue and red arrows point to the closed (oxidized) and open µs2L2 species respectively. Intracellular µs2L2 species purified from the murine hybridoma B1.8 were subjected to the sequential alkylation protocol described Intracellular µs2L2 species purified from the murine hybridoma B1.8 were subjected to the sequential alkylation and analyzed by mass spectrometry In the MS/MS spectrum Y and B ions are shown in blue and red, respectively.
Molecular and serological investigation of patient samples.Fig. 2molecular detection of nCoV-2019 in seven patients during two times of sampling. Patient information can be found in Extended Data Table 1 and 2. Details on detection method can be found in material and methods. BALF, bronchoalveolar lavage fluid; OS, oral swab; AS, anal swab. dynamics of nCoV-2019 antibodies in one patient who showed sign of disease on 2019.12.23 (ICU-06). For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls. serological test of nCoV-2019 antibodies in five patients (more information can be found in Extended Data Table 2). Star indicates data collected from patient ICU-06 on 2020.01.10. For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.
UBXN-3 stimulates disassembly of ubiquitylated CMG by CDC-48_UFD-1_NPL-4.Figure 4CDC-48, UFD-1_NPL-4 (U_N) and UBXN-3 (full-length and UBXN-3-∆UBX) were purified as described in Materials and Methods and analysed by coomassie blue staining of SDS-PAGE gels. Ubiquitylation and disassembly of the C. elegans CMG helicase was reconstituted in vitro as indicated (see Materials and Methods for further details). CMG was isolated by immunoprecipitation of SLD-5, after ubiquitylation in the presence of TIM-1_TIPN-1, before incubation with the indicated factors. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8. CMG disassembly was monitored by release of CDC-45 or MCM-2-7 proteins into the supernatant, whilst SLD-5 and other GINS subunits remained on the beads. Asterisks indicate non-specific bands in the anti-MCM-7 and CDC-45 immunoblots. Equivalent reactions to (C) involving ubiquityation of CMG in the absence of TIM-1_TIPN-1. For (C) and (D), the bands corresponding to MCM-7 conjugated to 1-5 ubiquitins are indicated in lane 4.
Fig.Figure 3a. Root growth rate (RGR in µm/min) of eir1-4 roots transferred 5 DAG to ammonium (grey) or nitrate (red) containing medium over a period of 680 minutes. Data represent the geometric mean (± standard error, SE) of 3 biological replicates (n=5 roots/condition). Light and dark periods are highlighted in light or dark gray. b and c. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG eir1-4 roots 12 HAT to ammonium (B) or nitrate (C) supplemented media. The first 20-20 epidermal and cortex cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (B) and in blue and green on nitrate (C), respectively. Scale bar=30 µm. Column bars denote the geometric mean of cell length at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 0.75884+0.02624 (ammonium, epidermis), 1.13088+0.08446 (ammonium, cortex) and 2.06912+0.10341 (nitrate, epidermis), 0.99878+0.07278 (nitrate, cortex). Data are derived from 3 biological replicates, n=9 (ammonium) and 8 (nitrate) roots. d. Average number of cell divisions along the epidermis (ep) and cortex (co) in 5 DAG old Col-0 and eir1-4 root tips 12 HAT to ammonium or nitrate supplemented media. Data are derived from n=15 and n=17 roots of Col and n=10 and n=9 roots of eir1-4 on ammonium and nitrate, respectively. Statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.
Figure 4(A) Immunohistochemistry of the spinal cords of AR-97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody. (B) Quantification of 1C2-positive motor neurons in the spinal cords (n = 5 per group). (C) Immunoblotting of the spinal cords of AR-97Q mice treated with or without RG108 for AR. (D) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR-97Q mice treated with DMSO or RG108 (n = 3). (E) Relative mRNA expression levels of human AR in AR-97Q mice spinal cord with or without RG108 treatment (n = 3). Immunohistochemistry for ChAT in the spinal cord anterior horns of AR-97Q mice (n = 5 per group). quantitative analysis of the neuron sizes in the spinal cord anterior horns of AR-97Q mice (n = 5 per group). (H,I) Western blot analysis (H) and relative signal intensities (I) of the ChAT-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3).
– Tau protein is necessary for the dendritic and synaptic maturation of newborn granule neurons.Figure 2A Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus. A schematic representation of Sholl´s analysis is shown in the WT image.B-C Quantification of total dendritic length (B) and Sholl´s analysis (C) of 4-week-old newborn granule neurons in WT and Tau-/- mice. D Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus.E-F Quantification of total dendritic length (E) and Sholl´s analysis (F) of 8-week-old newborn granule neurons in WT and Tau-/- mice. G Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).H-IQuantification of the number of PSDs/µm (H) and PSD area (I) in each dendritic branching order of 4-week-old newborn granule neurons in WT and Tau-/- mice. J Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).K-L Quantification of the number of PSDs/µm (K) and PSD area (L) in each dendritic branching order of 8-week-old newborn granule neurons in WT and Tau-/- mice.
Figure 5(a) Representative field of view showing Halo-TMRCTCF bound to pPlat-4xCTCF following 75 mM NaCl, 75 mM KCl buffer wash. Arrows denote CTCF bound at predicted site of 4xCTCF array. (b) Kymograph of salt-resistant Scc1GFP-TEV-cohesin failing to bypass DNA-bound Halo-TMRCTCF. DNA was post-stained with Sytox Green.
a) WB analysis shows accumulation of p62 in the liver of NRBF2 KO mice.f6(a) WB analysis shows accumulation of p62 in the liver of NRBF2 KO mice. Liver lysates were immunoprobed with antibodies indicated. p62 protein levels are upregulated in the KO mouse liver. (b) The quantification result shows that the p62 level increased more than threefold in the NRBF2 KO miceliver compared with WT miceliver. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test; WT, n=5 mice; KO, n=4 mice). (c) WB analysis shows accumulation of high-molecular weight (HMW) ubiquitin in the liver of NRBF2 KO mice. Insoluble fractions of liver lysates were immunoprobed with an anti-ubiquitin antibody, n=4 mice per genotype. (d) Atg14L-linked Vps34kinase activity is impaired in the mice of NRBF2 KO mice. Atg14L-linked Vps34 is pulled down by an anti-Atg14L antibody and is subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34kinase activity versus IPed Atg14L shows the significant difference between WT and NRBF2 KO miceliver. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=4 mice each group). (f) Focal accumulation of p62 in the liver of NRBF2 KO mice. Liver frozen sections (15 μm in thickness) were stained with an anti-p62 antibody. Focal accumulation of p62 is observed in the KO mice liver but not in the WT mice liver. Scale bars, 100 μm or 20 μm (amplified regions). This is a representative image from four mice per genotype. (g) Accumulated p62 colocalizes with NQO1. Liver frozen sections were double stained with an anti-p62 antibody and an anti-NQO1 antibody. This is a representative image from four mice per genotype. Scale bars, 20 μm.
Figure 5A CBLB first neighbors network within the CBL CN. Edge width is coded according to Pearson correlation coefficient (R<0.8 or R>0.8). The identified edges are depicted in red if they have been already reported as direct interaction in the BioGRID database. Proteins detected in the CBLB signalosome shown in Fig 3 are colored in red. B Histograms showing the number and corresponding percentage of CBL interactors also present in the CBLB signalosome according to their distance from CBLB in the CBL CN. The two depicted analyses correspond to the complete and to the high confidence (R>0.8) CNs are depicted. C Zoom on the high confidence (R>0.8) CRKL and CSK first neighbors subnetwork found within the CBL CN. The identified edges are depicted in red if they have been already reported as direct interaction in the BioGRID database. CSK and CRKL are highlighted in yellow. D, E, F, G, H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C. CD4+T cells were left unstimulated (-) or stimulated (+) with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates were incubated with isotype control (Iso Ctrl) or the specified antibodies and the resulting immunoprecipitates (IP) analyzed by immunoblot with the antibodies specified in the left margin. The validated PPIs predicted in C correspond to CSK-CBLB and CSK-CD5 (D), CRKL-PI3Kp85β and CSK- PI3Kp85β (E). Molecular masses are shown (kDa). Data are representative of at least 2 experiments. D, E, F, G, H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C. CD4+T cells were left unstimulated (-) or stimulated (+) with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates were incubated with isotype control (Iso Ctrl) or the specified antibodies and the resulting immunoprecipitates (IP) analyzed by immunoblot with the antibodies specified in the left margin. The validated PPIs predicted in C correspond to PI3Kp85β-PI3Kp110α, CSK-PI3Kp110α (F) CRKL-PI3Kp110α (G). Molecular masses are shown (kDa). Data are representative of at least 2 experiments. H Summary of the PPIs found in the high confidence CSK and CRKL first neighbors subnetwork (see C) that were validated by co-immunoprecipitation (see D to G). Interactions validated and non validated by co-immunoprecipitation are shown using red and blue edges, respectively. Interactions untractable by co-immunoprecipitation analysis due to the lack of specific antibodies are shown as gray edges.
Figure 5Representative coronal slices from lumbar spinal cord show the presence of both TMR-Tat-P4-(C5)2 and TMR-Tat-C5 peptides (magenta) 1 hour after i.t. administration in naïve mice. Scale bar 200µm. DH dorsal horn, VH ventral horn. Insert zooms of merged channels (right panels), scale bar 50µm. Representative immunoblots of co-immunoprecipited PICK1:GluA2 from spinal cord lumbar tract total lysates demonstrate partial disruption of this interaction 1 hour after 20 µM i.t. injection of Tat-P4-(C5)2. Densitometric analysis of immunoblots indicate a significant effect following Tat-P4-(C5)2 treatment, graph show mean +/- SEM and individual points, unpaired t-test, **p≤0.01, n=4/group Representative immunoblots of co-immunoprecipited PICK1:GluA2 from spinal cord lumbar tract total lysates demonstrates no disruption of this interaction 1 hour after 20 µM i.t. injection Tat-C5. Densitometric analysis of immunoblots indicate no significant effect of Tat-C5, graph show mean +/- SEM and individual points, unpaired t-test, p=0.10, n=4/group Representative immunoblots of lumbar spinal cord total lysates exhibit decreased pS880-GluA2 total levels following 20 µM Tat-P4-(C5)2, and Tat-C5 i.t. injections compared to saline injected animals. Densitometric analysis of immunoblot indicates a significant effect only for Tat-P4-(C5)2 (graph show mean +/- SEM and individual points, One-way ANOVA followed by Dunnett´s multiple comparisons test, ** p≤0.01, n=5-6/group as indicated by individual values) Surface biotinylation of spinal cord slices under basal condition demonstrates unchanged GluA1 and GluA2 surface level upon Tat-P4-(C5)2, Tat-C5 peptides incubation compared to the untreated condition (CTR). Densitometric analysis of immunoblots show that the reduction in mean surface for GluA2 in not significant (graph show mean +/- SEM and individual points, One-way ANOVA followed by Dunnett´s multiple comparisons test, n=5/group as indicated by individual values). Immunoblot of PICK1 following pull-down from lumbar spinal cord with Biotin-Tat-P4-(C5)2 or Biotin following 20 µM i.t. administration demonstrating in vivo target engagement.
Percentage of NDP52-positive S.figf4Percentage of NDP52-positive S. Typhimurium among ubiquitin-coated bacteria, determined by microscopy. HeLa cells transduced with the indicated NDP52 domain-deletion mutants fused to green fluorescent protein were infected with S. Typhimurium and stained for ubiquitin 2 h after infection. ZnF, zinc finger domain. *P 0.01, Student's t-test. Mean and s.d. of triplicate coverslips from two independent experiments; more than 50 ubiquitin-positive bacteria were evaluated per coverslip.
LAPTM4B inhibits EGF‐stimulated intraluminal sorting of EGFRMDA‐MB‐231 cells expressing GFP‐Rab5Q79L were starved and stimulated with 100 ng/ml Alexa‐555‐EGF for 90 min followed by fixation and immunostaining for LAPTM4B (blue).Figure 4MDA‐MB‐231 cells expressing GFP‐Rab5Q79L were starved and stimulated with 100 ng/ml Alexa‐555‐EGF for 90 min followed by fixation and immunostaining for LAPTM4B (blue). The percentages of luminal Alexa‐555‐EGF in individual endosomes were plotted against endosomal LAPTM4B levels. Trend lines and Pearson's correlation coefficients are shown. Data are representative for three independent experiments. C Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved overnight, cell surface EGFR was labeled with immuno‐gold on ice. Cells were then stimulated with EGF for 1 h at 37°C and fixed for the EM study. Scale bar, 200 nm. See Materials and Methods for details.D Relative amounts of immuno‐gold‐labeled EGFR in the MVE lumen versus MVE limiting membrane were quantified. Over 80 endosomes for each siRNA treatment from three independent experiments were used for quantification (mean + SD; ***P 0.001, one‐tailed t‐test). MDA‐MB‐231 cells were transfected with GFP‐Rab5Q79L and Flag‐LAPTM4B, starved, and stimulated with Alexa‐555‐EGF for 90 min and followed by intraluminal sorting analysis. See Materials and Methods for details. Quantification of EGF localization in LAPTM4B‐positive and LAPTM4B‐negative endosomes in (E) (mean + SD; n = 3; ***P 0.0007, one‐tailed t‐test).
Figure 5(a, b) The heat maps show the deconvolution scores of cell types across BALF samples grouped by diagnosis (a) and indicated clinical parameters (b). (c Empirical cumulative density plots depict the distribution of correlation coefficients for (c) Myofibroblast markers (red points) with DLCO in comparison to all background proteins (black line). d) Empirical cumulative density plots depict the distribution of correlation coefficients for (d) Plasma cell markers (red points) with % alveolar macrophages in BAL in comparison to all background proteins (black line). (e) Barplot displays coefficients derived from correlating lung function associations at the protein level with cell type specific ILD associations at the RNA level. (f) The heatmap illustrates gene expression changes associated with lung fibrosis across indicated cell types (columns) for selected BALF protein biomarkers (rows). The dotplot visualizes the frequency changes of the indicated cell types inferred from the deconvolution of bulk mRNA data of ILD samples compared to controls (GSE47460). Samples used in this study from the LTRC n = 254 ILD patients and n = 108 controls. (g) The protein features in BALF were used to train a random forest algorithm to predict lung function. The model was tested on transcriptional signatures from single cell RNA-seq data to correctly predict reduced lung function in end stage lung fibrosis when compared to controls. (h Box plots show predicted lung function changes (DLCO%) in the three single cell RNA-seq cohorts (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11) in (h) , i) Box plots show predicted lung function changes (DLCO%) in published bulk RNAseq of IPF samples from different histopathological stages (GEO GSE124685) (control n = 35, IPF1 n = 19, IPF2 n = 16, IPF3 n = 14) in (i).
Fig.Figure 4(A) De novo synthesized mitochondrial proteins were isolated after labelling with 35S-methionine for 1 hour. Positions of individual mitochondrial encoded proteins are indicated. (B) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin, doxycycline and chloramphenicol (+ antibiotic), followed by a 48 hours recovery period (- antibiotic). During both periods, proteins are isolated at 12h, 24h and 48h. Antibodies against subunits of C I (NDUFB6), C III (UQCRC1), C IV (COXI), MRPS35 and MRPL37 were used as indicated. (C) Quantitative analysis of the COXI recovery upon treatment with antibiotics (Actinonin, DOXY - doxycycline and CAP - chloramphenicol) as in B. The values represent ratios of recovered (48h, - antibiotics) versus untreated (0h) COXI levels. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4). (Actinonin and CAP n=3, DOXY n=4). (D) Sucrose density gradient sedimentation profile of ribosomal proteins and mtRNAs from heartmitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunobloting with antibodies against MRPS35 and MRPL12. The profile of a given mt-RNA is calculated after qRT-PCR analysis of individual fractions and corrected for its abundance in the cell according to results of Northern blot analysis. Results of individual experiments are provided for 12S and 16S rRNA, and for mt-mRNAs, results of distribution from two independent experiments using probes against ATP6, COXIII and ND5 were combined. Error bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=3). (E) Relative association of 12S rRNA, 16S rRNA and mt-mRNAs with mitoribosomes (55S) in Clpp knockout mitochondria comparing to wild type. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S and 16S n=3; mRNA n=6).
was analyzed in GEO data base.Figure 3A The relationship between ALKBH5 and TRAF4 was analyzed in GEO data base. Pearson's correlation analysis indicated TRAF4 and ALKBH5 had negative relationship (Cor = -0.70) B Western blot analysis of TRAF4 protein levels in control and ALKBH5-depleted cells after MDI-induced for 48 h and quantification of protein levels normalized to β-actin expression. C Western blot analysis of TRAF4 protein levels in control and ALKBH5-overexpressing cells after MDI-induced for 48 h and quantification of protein levels normalized to β-actin expression. D Western blot analysis of PPARγ and C/EBPα protein levels in control and TRAF4-depleted cells after MDI-induced for 48 h and quantification of protein levels normalized to β-actin expression. E Oil Red O staining of control, ALKBH5-depleted and ALKBH5+TRAF4 depleted cells after induced for 8 days. F qPCR analysis of PPARγ, C/EBPα and FABP4 expression in cell β-Actin was used as an internal control. G Western blot analysis of PPARγ and C/EBPα protein levels in cells , and quantification of protein levels normalized to β-actin expression.
siRNA depletion of CPAP abrogates the effects of CCB02 in extra centrosome containing cells (A, B) (i) Experimental strategy to test if the effect of CCB02 is CPAP dependent in MCF10A cells containing extra centrosomes (+Dox, extra centrosomes) (A) and MDA-MB-231 cells (B).Figure 4(i) Experimental strategy to test if the effect of CCB02 is CPAP dependent in MCF10A cells containing extra centrosomes (+Dox, extra centrosomes) (A Cells were treated with CPAP siRNA and control (scramble) siRNA for 48 hours. (ii) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAP Experimental strategy to test if the effect of CCB02 is CPAP dependent i MDA-MB-23 cell (B). Cells were treated with CPAP siRNA and control (scramble) siRNA for 48 hours. (ii) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAP (C) Fractions of cells containing centrosomes after siRNA treatment. 48 hours of siRNA treatment still contained a fraction of cells with extra centrosomes that were analyzed in the following experiments. Bar graph shows the percentage of cells with extra centrosomes in MCF10A (+Dox, extra centrosomes) in the presence of control siRNA or CPAP siRNA. Data represent mean ± SEM of three independent experiments. (N)=3, (n=300 cells per condition). p values were obtained using Ordinary One-way ANOVA test *P < 0.01, **P < 0.001 (D) Effect of CCB02 in the presence of control siRNA and CPAP siRNA in MCF10A (i) MDA-MB-231 cells (ii) CPAP depletion does not prevent centrosomes from clustering (second rows of i and ii). Note, in CPAP depleted cultures, centrosomes are marked with Cep152 (green) where CPAP (magenta) is detected faintly. In CPAP depleted cells, CCB02 does not prevent extra centrosomes from clustering (fourth rows of i and ii). Note CCB02 does prevent extra centrosomes from clustering in cultures treated with control siRNA (third rows of i and ii). Cells are stained for Cep152 (green), CPAP (magenta), microtubules (α-tubulin, red) and DNA (DAPI, blue). Scale bar, 2µm (E) Bar graph shows the percentage of cells with multipolar mitosis. Higher percentage of multipolar mitotic cells is observed in control siRNA-treated cultures. Bar graph on right shows the percentage of cells with multipolar mitosis. Data represent mean ± SEM of three independent experiments. (N)=3, (n=680 cells). p values were obtained using unpaired t test. ***P < 0.0001 (F) Bar graph shows the percentage of living (TUNEL-negative) and dyeing (TUNEL-positive) cells under various conditions tested. Higher percentage of TUNEL-positive cells is observed in CCB02-treated cultures. Data represent mean ± SEM of three independent experiments. (N)=3, (n=500 cells per condition). p values were obtained using Ordinary One-way ANOVA test *P < 0.01.