EMBO/sd-character-level-ner huggingface dataset:
a) Western blot analysis of parental SH-SY5Y cells treated with CCCP for the indicated times.figf7(a) Western blot analysis of parental SH-SY5Y cells treated with CCCP for the indicated times. Time-course experiments of CCCP-treated neuronal cells reveal increasing modifications of VDAC1 over time. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. All proteins represent endogenous levels. (b) Immunoprecipitations of Parkin and VDAC1 from parental SH-SY5Y cells confirm ubiquitylation of both proteins. CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time. Using specific antibodies, Parkin and VDAC1 were immunoprecipitated from lysates of parental SH-SY5Y cells at different time points, western blotted and probed with anti-ubiquitin, anti-Parkin and anti-VDAC1 antibodies. (c) Pathogenic Parkin mutations fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. Western blot analysis of a time-course experiment with inducible Parkin SH-SY5Y cells after CCCP treatment. Total lysates were prepared, and western blots were probed with specific anti-Parkin and anti-VDAC1 antibodies. β-Actin probing served as a loading control in a-c. WT, wild-type; Ub, ubiquitin.
Figure 3(A) Definition of two different ZEB1 peak subsets based on the presence or absence of overlapping YAP and JUN peaks. The two illustrative genome browser images show one example of a ZEB1-only peak and one example of a ZEB1/YAP/JUN peak (at the EFEMP1 gene and within the SHROOM3 gene, respectively). Control (ctrl) is the input for ZEB1 and IgG for the YAP and JUN ChIP-seqs. ChIP-seqs and respective controls were scaled accordingly. (B) Circle plot displaying the genome-wide distribution of ZEB1-only (middle circle, blue) and ZEB1/YAP/JUN peaks (inner circle, orange) across all chromosomes (outer circle). Peak density is visualised as a heatmap with more intense colours indicating higher accumulation of peaks at a specific location. (C) Localisation of ZEB1 peak summits relative to the closest TSS. (D) Distribution of ZEB1-only and ZEB1/YAP/JUN peaks in respect to different genomic features. Promoters were defined as 1.5 kb upstream to 0.5 kb downstream of the TSS. Promoter peaks were annotated to their respective genes and integrated with transcriptome data of ZEB1 knockdown compared to control MDA-MB-231 cells. (E) Hypergeometric testing showed that the association of ZEB1-only promoter peaks with genes repressed in the presence of ZEB1 is stronger than with activated genes, while ZEB1/YAP/JUN promoter peaks are only associated with activated genes. Odds ratio (OR): measure of association between categorical variables with OR > 1 indicating a positive association. (F) GO term analysis of the ZEB1-only repressed and ZEB1/YAP/JUN activated gene sets using Enrichr. Odds ratios are given in brackets.
a) Identification of Beclin1 as a substrate of ROCK1 via affinity purification.f1(a) Identification of Beclin1 as a substrate of ROCK1 via affinity purification. Left panel: HeLa cells growing in control (C: nutrient-rich media) or starved in HBSS (H) for 4 h were lysed and subjected to IP with ROCK1 cross-linked beads. Proteins bound were eluted and detected by SDS-PAGE, followed by silver staining that presents precipitated proteins identified by LC-MS/MS. Right panel: HeLa cells grown in control media or HBSS (4 h) were collected, whole cell lysates prepared and immunoprecipitated with ROCK1 cross-linked beads. The resulting immune complexes were divided into two, and were subjected to SDS-PAGE and analysed for Beclin1 (MES buffer) and ROCK1 (MOPS buffer). Inputs and IP were analysed by western blotting. HBSS-treated cell extracts were used for IgG IP controls. Whole cell lysates (10%) were run for inputs. Total cell lysates were rerun and analysed for LC3 and β-actin. (b) Co-IP was performed with 293T whole cell extracts by using ROCK1-crosslinked agarose, and the resulting IP was blotted with indicated antibodies. 293T cells transfected with Flag-Beclin1 for 36 h, cultured in control media or starved (4 h), were immunoprecipitated with anti-Flag beads. Inputs and immune complexes were resolved and detected. Total cell lysates were rerun and examined for LC3 and β-actin expression. (c) Interaction of YFP-ROCK1 and CFP-Beclin1 was examined by using live cell spinning disk microscopy and FRET in live cells. Graph represents FRETAverage ratio changes (YFP-ROCK1/CFP-Beclin1) upon starvation. Data represents mean±s.e.m.; n=6. Three different experiments were performed. For the control, the right top graph represents CFP intensity over time (corrected for photobleach), and the right bottom right represents YFP intensity (corrected for photobleach). In both control FRET experiments, no significant change was observed in fluorescence, in the presence or absence of HBSS medium. P values were calculated using Student's t-test.
Figure 2(A) DABimmunohistochemistry staining of Fstl1 with ischemic heart at day 3 after LAD ligation. Arrows indicate a subset of Fstl1 positive cells. Counter staining was performed with hematoxylin. Scale bar indicates 50μm. (B) Immunofluorescent microscopy staining of Fstl1 (green) and sarcomeric actin (red). Fstl1 protein was detected in interstitial areas of the ischemic and border zone but not in cardiomyocytes. Scale bar indicates 100μm. (C) Immunofluorescent microscopy staining of Fstl1 (green) and S100a4 (red) in the ischemic area. Arrows indicate Fstl1 and S100a4 double positive cells. Scale bar indicates 20μm. (D) Immunofluorescent microscopy staining of Fstl1 (green) and α-smooth muscle actin (red). Arrows indicate Fstl1 and α-SMA double positive cells. Scale bar indicates 20μm. Nuclei were stained by DAPI.
Figure 6Cultured astrocytes engulf microglial debris. CAG-EGFP mouse-derived primary astrocytes were co-cultured with or without fluorescence-labelled apoptotic microglia. (A) Fluorescence images of GFP (green), microglial debris (red) and GFAP immunoreactivity (blue). Images were acquired using the same laser power and sensitivity, and image processing was the same in the two images (red). Scale bar, 50 µm. (B) Fluorescence images of GFP (green), microglial debris (red) and LAMP-1 immunoreactivity (cyan). Scale bar, 30 µm and 5 µm (inset). C Flow cytometric analysis of uptake of microglial debris by cultured astrocytes. Fluorescence of internalized microglial debris was measured in GLAST+ astrocytes co-cultured with or without fluorescence-labelled apoptotic microglia. D Expression of TAM family members and their knockdown by siRNA in cultured astrocytes. Tyro3, Axl and Metk in cultured astrocytes or hippocampal tissue were examined by western blotting. Arrows indicate the corresponding protein band(s). The secondary antibody against goat IgG generates a ~115 kDa non-specific band. The ~115 kDa band in the Axl image is the Axl-specific band overlapping the non-specific band. The ~115 kDa band in the Mertk image is the non-specific band. Axl and Mertk expression was knocked down by two siRNAs. GAPDH expression is shown as an internal control. E Quantification of siRNA knockdown in cultured astrocytes (n = 3 independent experiments, one-way ANOVA with repeated measures). Knockdown of Tyro3 mRNA was examined by qPCR because of the very low expression level even in control astrocytes. Knockdown efficiency of Axl and Mertk was quantified from western blots. Results are normalized to Gapdh or GAPDH, and are shown as ratios to the value of non-transfected astrocytes. F, G Flow cytometric analysis of microglial debris uptake by cultured astrocytes. After knockdown of TAM family members, astrocytes were co-cultured with fluorescence-labelled apoptotic microglia. Fluorescence of internalized microglial debris in GLAST+ astrocytes was quantified by flow cytometry (n = 4 independent experiments, one-way ANOVA with repeated measures). Results are shown as ratios to the value of non-transfected astrocytes.
Figure 4(A) Micrographs of in situ hybridization for miR-204 in an adult mouse brain section. (A' and A'') are magnifications of boxed areas in (A or A'), respectively. (B) Dot plot showing miR-204 levels in ChP and SEZ in the adult mouse brain measured by RT-qPCR. (C) Agarose gel of RT-qPCR product loaded after the saturation phase showing presence of miR-204 and U6 in mouse ChP, SEZ and CSF. Plots depicting the number and size of EVs isolated from human CSF (D) Plots depicting the miR-204 levels (E) in EVs and EV-free CSF (n=2). (F) Agarose gel of RT-qPCR analysis depicting levels of miR-204 in the EV-free supernatant and EV containing fraction of human CSF loaded at the saturation phase. (G) Micrographs of transmission electron microscopy imaged extracellular vesicles (EVs) isolated from sham treated (upper row) and GW4869 treated animals (lower row). (G'' and G''') are magnifications of boxed areas in overview images to the left. (H) Dot plot depicting miR-204 levels in the CSF isolated 2 h after the ventricular injection of GW4869 inhibitor.
a,b) Body weight (a) and visceral fat mass (b) isolated from 2-month-old-mice with the indicated genotypes.figf6(a,b) Body weight (a) and visceral fat mass (b) isolated from 2-month-old-mice with the indicated genotypes. n = 5 mice per group. (c-i) Serum metabolic profile in HDad-TFEB mice compared with control mice. (j,k) Respiratory exchange ratio (RER; VCO2/VO2; j) and fatty acid utilization calculated from data in j (k). Values are mean ± s.d (n = 10 mice per group) *P≤0.05;**P≤0.01; compared with controls. VLDL, very-low-density lipoprotein; HDL, high-density lipoprotein.
Fig.Figure 4A. Immunofluorescence images of 10 μm-thick sections of CP from third ventricle villi of Mpdz+/+ and Mpdz-/- mice were immunolabeled as shown. The areas in the square frames are magnified in the bottom panels. CP sections from lateral ventricle villi immunolabeled for ZO1 (B) The images are representative of 2 Mpdz+/+ mice and 3 Mpdz-/- mice. Scale bars, top panels, 100 μm; bottom panels, 10 μm. CP sections from lateral ventricle villi immunolabeled for E-cadherin (E-cad; C). The images are representative of 2 Mpdz+/+ mice and 3 Mpdz-/- mice. Scale bars, top panels, 100 μm; bottom panels, 10 μm. D. Immunoblots with the indicated antibodies (top) and their quantifications. The densitometry measurements were normalized relative to the signal of the samples transduced by non-targeting shRNA (Ctrl). Note that the same β-actin immunoblot was used twice because the Jam-C and E-cadherin samples were immunoblotted on the same membrane (mean ± SD, n=3).
Figure 1Box-plot showing the enrichment scores (E-scores) of all possible 8-mers containing the G-box (CACGTG), PBE-box (CATGTG), and BRRE elements (CGTGC/TG). Boxes represent the 25-75% quartiles and the black line the median of distribution. Bars indicate the 1-25% (above) and 75-100% (below) quartiles. E-scores above 0.4 denote that binding of the proteins towards the indicated DNA element is statistically significant. Dashed blue line indicates the 0.4 threshold Sequence logo representation of the top scoring 8-mers obtained by hybridization with the PIF4, BES1 and PIF4-BES1 proteins Protein-binding microarrays (PBM) were probed with PIF4-His, MBP-BES1 and the PIF4-His/MBP-BES1 complex. For PIF4-BES1 complex purification, both proteins were co-expressed in E. coli using the double expression pCOLADuet-1 vector. An anti-MBP antibody was used for BES1 and BES1-PIF4 signal detection, while PIF4 was detected with an anti-His antibody Electrophoretic mobility shift assays (EMSA) showing interaction of the PIF4, BES1, and PIF4-BES1 proteins with the conserved G-box, PBE-, and BRRE-elements in the PIL1 (At2g46970), PRE5 (At3g28857), and DWF4 (At3g50660) promoters. Increasing amounts of protein were used for the assay Protein-binding microarrays (PBM) were probed with PIF4-His, MBP-BES1 and the PIF4-His/MBP-BES1 complex. For PIF4-BES1 complex purification, both proteins were co-expressed in E. coli using the double expression pCOLADuet-1 vector. An anti-MBP antibody was used for BES1 and BES1-PIF4 signal detection, while PIF4 was detected with an anti-His antibody BES1 binds both BRRE- and G-box elements as a homodimer. A deletion of BES1 (delN) fused to MBP (MBP-delN) and the complete protein (MBP-BES1) were co-expressed in E.coli. Formation of intermediate mobility bands, indicative of DNA recognition by a dimeric form of the protein was tested in EMSA assays. A signal corresponding to the dimeric full-length MBP-BES1 and MBP-delN complex was detected with both DNA probes. Increasing amounts of the proteins were used in the assay Co-expression of the PIF4 and BES1 effector constructs leads to synergistic pPIL1 activation, and reverses BES1-dependent inhibition of the pDWF4 reporter. The pPIL1 and pDWF4 promoters including three G-boxes (green boxes) and two BRRE- elements (orange boxes) were fused to the firefly luciferase reporter gene (LUC) and co-transfected with 35S::PIF4, 35S::BES1 and 35S::bes1-D effector constructs into Nicotiana benthamiana leaves. Leaf discs were collected 48 hours after infiltration and luciferase activity was measured in a microplate luminometer.
Figure 3(A) Relative reactive oxygen species (ROS/mg protein) production in control (CTNSWT), CRISPR-generated cystinotic cells (CTNS-/-), and patient-derived cystinotic (CTNSPatient) cells under fed condition (n = 3). (B, C, D) ROS production (ROS/mg protein) in CTNSWT, CTNS‑/-, and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG (4 hrs), respectively (n = 3). E, Viability test in starved (-AA) CTNSWT, CTNS‑/-, and CTNSPatient cells in the presence or absence of DMKG (2 mM) for 24 hrs. Results are shown relative to the fed condition (n = 3). Representative confocal micrographs (scale bars are 20 µm) of caspase 3/7 activation in DMKG (2 mM)-treated CTNSWT, CTNS‑/-, and CTNSPatient cells for 24 hrs (n = 3). Representative immunofluorescence analysis of caspase 3/7 activation in DMKG (2 mM)-treated CTNSWT, CTNS‑/-, and CTNSPatient cells for 24 hrs (n = 3). (H, I) Western blotting and densitometric analyses for LC3-II/LC3-I ratio in CTNSWT, CTNS‑/-, and CTNSPatient cells cultured in the presence or in the absence of BafA1 (25 nM) and DMKG (2 mM) for 4 hrs, respectively (n = 3).
Figure 2(A) PD experiment with biotinylated peptides of H4 (1-23aa) and total lysates of HCT-116 cells. (B) Quantification of the IκBα 60 kDa band (upper panels) and the 37 kDa band (lower panels) is shown as average and s.d. of three independent experiments. (C) PD experiments with GST-SUMO IκBα fusion protein and serial dilutions of histone-enriched HEK-293T lysates. Quantification of three independent replicates is shown. (D) Venn diagrams representing the overlap between IκBα and acetylated H4 peaks obtained in ChIP-seq experiments from HCT-116 cells. (E) Peak distribution of acetylated H4, H4K12ac and total histone H4 obtained in IκBα target genes and non-IκBα targets relative to the transcription start site (TSS).
Figure 5A. Targeted deletion of nucleolin GAR domain by CRISPR-Cas9. Schematic shows mouse nucleolin exons targeted by single guide RNAs (sgRNAs) and resulting deletion in the GAR domain amino acid sequence. B. Western blot analysis of nucleolin in DRG neurons from wildtype (WT) and GAR+/- mice cultured in Boyden chambers. Tuj1 was used as a loading control. The higher nucleolin band in GAR+/- corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion (corresponding to about 4 kDa reduction in protein size).
Western Blot analysis with LC3B-antibody in ASPC-1 and MiaPaCa-2 cells.Figure 9  The experiment was repeated three times with nearly identical results. The LC3-I fractions can be clearly distinguished above the LC3-II signals. Bafilomycin A1 elevated slightly the LC3-II signal intensity compared to control in both cell lines. There was a marked and dose-dependent increase of the signal strength of both fractions when OMP was used. 5-FU alone did not relevantly influence the LC3-level. The corresponding β-Actin level is shown below the LC3-WB to confirm the correctness of this semiquantitative evaluation.
Dysregulated NFκB dynamics diminish the cellular discrimination of TNF exposures (A) Simulations of A20 mRNA concentrations in versions of the NFκB-necroptosis model where expression is under the control of synthetic NFκB activity following step functions of 0.Figure 4(A) Simulations of A20 mRNA concentrations in versions of the NFκB-necroptosis model where expression is under the control of synthetic NFκB activity following step functions of 0.5, 1, 2, 4, 8, or 16 hours duration (smoothed line is population average, and shaded area the 30th percentile around the median). (B) Fractional survival that results from simulations in (A). (C) Immunoblot for IκBα and IκBε in L929 wildtype and CRISP/Cas9-knockout cell lines. Asterisks depict unspecific bands. (D) Normalized RelA activity dynamics after TNF treatment quantified via EMSA (mean of three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05, **P<0.01; corresponding images of representative experiment in Figure EV4A). (E) A20 mRNA quantified via qPCR (mean of three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05). (F) Immunoblot for A20 (representative data of three independent experiments). (G) Fractional survival after 24 hours of TNF treatment in indicated cell lines (mean of three independent experiments ± standard deviation; two-tailed Student's t-test **P<0.01). (H) Death rates in TNF-treated indicated cell lines including isogenic IκBα/IκBε-knockout population (mean of three independent experiments ± standard deviation). (I) Death rates in TNF-treated cell lines treated with targeting (siA20) or non-targeting (siCon) siRNA (mean of three independent experiments ± standard deviation).
Systemic autoimmune responses in mice lacking secondary lymphoid organs3‐month‐old wild‐type (C57BL/6), Ltbr−/−, Lta−/−, LIGHT−/−, and Ltbr−/−Hox11−/− mice; percentage of mice with at least one ANA.Figure 13‐month‐old wild‐type (C57BL/6), Ltbr−/−, Lta−/−, LIGHT−/−, and Ltbr−/−Hox11−/− mice; percentage of mice with at least one ANA. 6‐month‐old Ltbr−/− mice (n = 15); titers of individual mice. LIA of 6‐month‐old Ltbr−/− mice, wild‐type mice, and 3‐month‐old Rorγt‐Ltb−/− mice. LTβR‐Fc or control immunoglobulin (Ig)‐treated wild‐type mice; percentage of mice with at least one ANA. E denotes gestational day. *ANA were determined with LIA at the age of 3 months. **ANA were determined with LIA 3 months after the start of treatment. 3‐month‐old cell‐specific LT knockout mice; percentage of mice with at least one ANA. Bone marrow cells from wild‐type or Ltbr−/− mice were transferred at neonatal age into lethally irradiated wild‐type or Ltbr−/− mice; percentage of mice with at least one ANA. ANA were determined with LIA at the age of 3 months.
Figure 4(A) Decreased BIK1 abundance in map4k4-1. 35S::BIK1-HA was introduced into map4k4-1 by crossing, sibling lines carrying either map4k4-1 or MAP4K4 genotype were isolated from F3 generation. BIK1 levels were detected by western blot analysis using anti-HA antibodies. (B) MAP4K4 regulates BIK1 accumulation through the proteasome pathway. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with DMSO (-) or the proteasome inhibitor MG132 (50 µM) for 5h, then the plants were collected for immunoblot analysis. (C) Flg22-induced BIK1 phosphorylation assay. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with H2O (-) or 1 µM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot. (D, E and F) Identification of phosphorylation sites by LC-MS/MS analysis. Five residues in GST-BIK1-K105E were phosphorylated in vitro by HisMBP-MAP4K4. The peptide sequences are shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions. (G) S233 and T242 were required for BIK1 hyper-phosphorylation. BIK1-HA, BIK1S233-HA and BIK1S242-HA mutants were expressed in Col-0 protoplasts and treated with 100 nM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot. Each experiment was performed at least three times with similar results.
Fig.Figure 7HEK293 cells were transfected with α11.2, α2δ-1, and β2A before whole cell patch recording in 20 mM Ba2+. Representative current traces of the first 2 ms obtained from recordings upon depolarizations from a holding potential of -80 mV to the indicated potentials (the voltage protocol is schematized in the upper left corner). Representative current traces upon step depolarizations to the reversal potential (Erev) for 20 ms to obtain movement of the ON-gating charges (Qon), and subsequent to -50 mV for 10 ms to obtain tail currents (Itail). Insets: magnifications of exemplary Qon for CaV1.2 WT, K1647A, Y1649A, I1654A, F1658A and K1662E. Plots of Itail (in this panel corrected for variations in cell capacitance) versus total detectable charge transfer for Qon. Slopes of regression curves are strongly reduced for CaV1.2 K1647A, Y1649A and I1654A versus WT (see Table 7 for more details). Dot plots and means + SEM of Qon (in this panel corrected for variations in cell capacitance; *p<0.05; **p<0.01; one-way ANOVA with Bonferroni post-hoc test; n = 13-18; see Table 7 for more details). The reduction in slope of the regression curve of combined population data for K1647A, Y1649A and I1654A versus WT indicates reduced coupling of Itail with Qon when α-actinin binding to the IQ motif is diminished.