EMBO/sd-character-level-ner huggingface dataset:
a) Purified autolysosomes were treated with trypsin.figf7(a) Purified autolysosomes were treated with trypsin. The trypsin-treated (stripped) autolysosomes were incubated with rat brain cytosol and reaction mixture, and then analysed by western blotting. HC, heavy chain. (b) Scanning electron micrographs of purified autolysosomes, rat brain cytosol, trypsin-treated purified autolysosomes and trypsin-treated purified autolysosomes after the membrane budding assay. Scale bar, 250 nm. The arrows indicate buds. (c) Trypsin-treated autolysosomes were incubated with rat brain cytosol and reaction mixture in the presence of antibodies against GFP, clathrin, AP2, AP3, AP4 and PtdIns(4,5) P2, and then analysed by western blotting. (d) Reactions from c were visualized by scanning electron microscopy. Scale bar, 250 nm. Arrow indicates bud. (e) Samples from d were quantified for the number of buds on each autolysosome. Error bars represent s.d. n = 50 autolysosomes from three independent experiments. Uncropped images of blots are shown in Supplementary Fig. S6.
promotes proteasome recruitment to polysomesA.Figure 6A. Trypsin-like proteasome activity determined in fractions as described in Fig. 4A using an in vitro assay. Results are shown relative to the proteasome activity obtained in MCF7 cells maintained at 37 oC (means +/- SEM, n = 4). B. MCF7 cells were exposed to the proteasome inhibitor MG132 (20 μM) or 42 oC for 1h after which an equal amount of cell lysate was spun through a 30% sucrose cushion. Resulting polysomes were assayed for the levels of 26S proteasome subunits and HSPA1. C. Cell lysates from MCF7 cells maintained under the conditions indicated were fractionated by sucrose density gradient centrifugation. Polysomal fractions were subjected to immunoprecipitation with RPS19 antibody, and precipitated were analyzed by quantitative LC-MS/MS. The heat map represents normalize spectral abundance factors (NSAF). D. Total cell lysates were prepared from naïve MCF7 and thermoresistant MCF7aTT cells, and puromycin labeled peptides were pull down as illustrated in the scheme. Precipitates were assayed for the presence of K48-linked polyubiquitin, chaperones, and proteasome subunits by immunoblotting. E. Partitioning of the 20S proteasome subunit PSMB1 along the sucrose gradient under the indicated conditions was assessed by immunoblotting, black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity. The line graph on the top shows trypsin-like proteasome activity in the same fractions (means +/- SD, n = 3).
Monitoring co-translational membrane protein insertion in real time by FRET.Figure 1Expected FRET changes during co-translational insertion of an N-out membrane protein. The FRET acceptor (Atto655, red star) was placed at the N-terminus of the nascent chain, the FRET donor (Atto488, yellow star) at one of two positions in SecY (see panel B). Positions on SecY (cyan) used for site-specific Atto488 donor labeling at the cytoplasmic face (position 111; red sphere) or periplasmic face (position 212; black sphere). (SecE, orange; SecG, green). The SecYEG translocon is embedded into phospholipids contained in a nanodisc held together by membrane scaffold proteins. Model based on PDB ID: 3J45 . Time-dependent acceptor fluorescence changes due to FRET during co-translational insertion of acceptor-labeled LepB75 into donor-labeled translocon.
Normal skeletal muscle morphology in hibernating squirrels.Figure 1A. Left column, an active summer squirrel; right column, a torpid squirrel. B. The morphology of quadriceps is unchanged by hibernation as seen in haematoxylin and eosin (H&amp;amp;E) stained sections (scale bar 90 µm). C. Dystrophin staining was performed to outline the sarcolemma to determine percentage distribution of minimum Feret's diameter. D. Average ± SD of minimum Feret's diameter in quadriceps (p = 0.26) and tibialis anterior (p = 0.33) muscles is not significantly different between summer and hibernation.
Figure 7(A) Model of potential Gata6 role upstream of Edaradd/NF-B. (B) qRT-PCR confirmation of the change in Edaradd mRNA expression in-vivo from mouse whole skin (average SD, 1d n=3, 2d n=4). *Unpaired t-test P-values: 1d = 0.02, 2d = 0.05. Mann-Whitney U test P-values: 1d=0.06, 2d=0.03. (C) ChIP-qPCR for Gata6 at three conserved binding sites within the Edaradd promoter. GAPDH served as a negative control. Binding site 1, 2, and 3 are located 50 bp, 1 kb, and 1.5 kb upstream of the transcription start site, respectively (average SD, n=4). *Unpaired t-test P-values GAPDH=0.4, 1=0.002, 2=0.8, 3=0.2. Mann-Whitney U test P-values: GAPDH=0.3, 1=0.06, 2=0.9, 3=0.2. (D) Western blot from whole skin lysate. Note decrease of Edaradd and activated pNF-B in the iKO. (E-F) Staining for Edaradd and quantification show decrease in Edaradd protein levels in the matrix of Gata6 iKO. *Unpaired t-test P-value = 3.5x10-14. n=100 follicles from 3 mice per genotype. (G-H) Staining for pNF-B and quantification show decrease of active NF-B in the Gata6 iKO matrix. *Unpaired t-test P-value = 2.2x10-16. n=50 follicles from 3 mice. (I) Rescue of Gata6 iKO keratinocyte growth by transfection with pGata6 or pEdaradd. Cell plates of keratinocytes are stained with Rhodanile Blue (n=3 cell lines each). (J) Quantification of H2A.X in cells stably transfected with pMock, pGata6, or pEdaradd plasmids, followed by induction with TM for 6 hours (average SD, n=4 with > 500 cells counted per condition). *Unpaired t-test P-values = 2x10-4 (iKO pGata6 vs pMock), 2x10-4 (iKO pEdaradd vs pMock). (K) qRT-PCR analysis of Mcm10 expression in pMock, pGata6, and pEdaradd WT and iKO expressing cells (average SD, n=4). *Unpaired t-test P-values: 8x10-4 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock). Mann-Whitney U test P-values: 0.03 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock).
Figure 5(A, B) Co-immunoprecipitation of ectopically expressed Myc-CDC20 171-499 fragment (containing WD40 domain) (A) rather than Myc-CDC20 1-170 fragment (lacking WD40 domain) (B) with endogenous p65 in HEK293T cells. (C) Schematic representation of RHD domain location in p65 peptides (amino acids 19-306). (D) Western blot analyses of Flag-p65, Flag-p65 1-310 fragment (containing RHD domain), Flag-p65 311-551 fragment (lacking RHD domain) plasmids expression in HEK293T cells. (E, F) Co-immunoprecipitation of ectopically expressed Flag-p65 1-310 fragment (containing RHD domain) (E) rather than Flag-p65 311-551 fragment (lacking RHD domain) (F) with endogenous CDC20 in HEK293T cells. (G, H) Co-immunoprecipitation of ectopically expressed Myc-CDC20 171-499 with ectopically expressed Flag-p65 1-310 fragment in HEK293T cells.
Fig.Figure 4A) Synthetic reporter proteins carrying EGFP either at the lumenal or cytosolic end of the single-spanning transmembrane domain of Nyv1, which carries no hydrophilic extensions at its lumenal C-terminus. B) The topology of these constructs in the vacuolar membrane is indicated. C,D) FRAP assays for fusion pores. The indicated cells expressing (C) yeGFP-LT or (D) LT-yeGFP were labeled with FM4-64 and subjected to FRAP experiments Cells were imaged before and 0 (bleached) or 30 seconds after photobleaching (recovery). The bleached areas are indicated by an arrow. E,F) FRAP assays for fusion pores were performed as in (C, D), but with nyv1∆ cells. Scale bar: 2 µm. G) The histogram reports the fraction of cells showing FRAP. Means and s.d. are shown from 3 independent experiments, with 100 cells analyzed in each.
Figure 1A. Relative levels of OMA1 mRNA in colorectal cancer tissues (n=105) compared with adjacent normal tissues (n=43) from colorectal cancer patients were shown (using the GEO dataset GSE21510). The data are presented as mean ± SEM, and statistical significance was determined by a Mann-Whitney test. ***p < 0.001. B. Kaplan-Meier curves were constructed to analyze and compare between patients with high and low levels of OMA1 in colorectal cancer samples from the GEO dataset GSE17537. "Low" indicates patients with OMA1 mRNA levels less than the median. "High" indicates patients with OMA1 mRNA levels greater than the median. Statistical analysis was performed using log-rank tests, n = 55, p=0.0180. C. The lysates of tumors (T) and adjacent normal (N) tissues from colorectal cancer patients were analyzed by Western blotting with antibodies against OPA1 or Tubulin. The "c, d and e" bands of OPA1 indicate cleaved OPA1 bands. Tubulin was used as a loading control. D. Control and OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0 (normoxia), 24, or 48 hours. Cell lysates were then assessed by Western blotting with antibodies against OPA1, OMA1, or β-Actin. β-Actin was used as a loading control. Representative immunoblots were from n = 3 independent experiments. E. OPA1-null mouse embryonic fibroblasts (MEFs) expressing control (empty vector) or OPA1 isoform 1 were cultured in normoxia or hypoxia (1% O2) for 24 hours. Cell lysates were analyzed by Western blotting with anti-OPA1 or anti-Tubulin antibody. Representative immunoblots were from n = 3 independent experiments. F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were cultured in hypoxia (1% O2) for 0, 24, or 48 hours, and the cell lysates were assessed by western blot with antibodies against OMA1, and GAPDH. β-Actin was used as a loading control (representative data from three independent experiments). The asterisk indicates a nonspecific band. G. The relative protein levels were evaluated by densitometry analysis using ImageJ software and were quantified for the ratio of OMA1-Flag/β-Actin or E324Q-Flag/β-Actin in hypoxia for 0, 24 or 48 hours (n = 3 independent experiments). The data are presented as mean ± SD.
Figure 5(A) Regime of the mouse experiment. Balb/c mice were a mouse-adapted strain of pandemic H1N1 viru. At 24 hours before inoculation, 8, 24 and 36 hours post-inoculation, two groups of mice were intranasally administered with wtSPINK6 protein or mutSPINK6 protein. Ten mice treated with wtSPINK6 or mutSPINK6 were monitored daily for disease signs, body weight and survival for 14 days. Five mice treated with wtSPINK6 or mutSPINK6 were sacrificed at 3 and 4 days after the viral challenge. Lung tissues were collected for the quantification of viral growth and immunofluorescence staining. (B) Survival rates of mice treated with wtSPINK6 and mutSPINK6 was analyzed with Mantel-Cox test. (C) Viral load and viral titer in the lung homogenates of mice treated with wtSPINK6 and mutSPINK6. Data represent mean ± SD. **, P < 0.01; ***, P< 0.001. Student's t test. (D) Mouse lung tissues are applied to immunofluorescence staining to identify the viral NP positive cells (green). Nuclei are counterstained with DAPI (blue). Representative confocal images of virus-infected cells in the indicated mice. Scale bar, 10 µm.
Figure 9A. Overexpression of cortactin compensated for the knockdown of KV10.1 in ciliary disassembly (4 h serum reintroduction after starvation). B. Deletion of the domain of KV10.1 responsible for interaction with cortactin abolished the effects of KV10.1 overexpression on the abundance of cilia. C. In mouse embryonic fibroblasts, active (phosphorylated) cortactin is increased as compared to wild type, possibly compensating the lack of KV10.1.
a) Schematic representations of all constructs tested.figf2(a) Schematic representations of all constructs tested. R, mRFP; RH, mRFP fused to HSC70bm (VKKDQ and KFERQ); RS, mRFP fused to scrambled QBP1 (Online Methods); RHS, mRFP fused to HSC70bm and scrambled QBP1; RQ, mRFP fused to QBP1; RHQ, mRFP fused to HSC70bm and QBP1 (Sc, scrambled). (b) Confocal images of the 150Q Neuro2a cells transfected with R, RH, RS, RHS, RQ and RHQ. Molecules containing QBP1 co-localized with the tNHTT-150Q-EGFP inclusions (bar, 5 μm). Cells with inclusions were chosen to illustrate the co-localization of QBP1-containing molecules with tNHTT-150Q-EGFP. (c) The expression of RQ and RHQ in 150Q Neuro2a cells decreased the accumulation of insoluble tNHTT-150Q-EGFP on the gel top. Note the shift of tNHTT-150Q-EGFP from an insoluble to a soluble form when RQ was co-expressed. The levels of tNHTT-16Q-EGFP were not affected. Full-length western blots are presented in Supplementary Figure 13. (d) RHQ reduced the levels of aggregated and soluble forms of tNHTT-150Q-EGFP by 81.8% and 33.6%, respectively. WB, western blot. (e) Quantification of tNHTT-16Q-EGFP protein levels. (f) ArrayScan analysis revealed 28% and 56% reductions in inclusion formation in 150Q Neuro2a cells by RQ and RHQ, respectively, after 24 h of differentiation and induction. (g) In 150Q-nls Neuro2a cells, expression of RQ and RHQ reduced the number of tNHTT-150Q-nls-EGFP inclusion by 23% (RQ) and 53% (RHQ) after 48 h of differentiation and induction compared to RS and RHS, respectively. Bars in d and e represent mean values ± s.e.m. from four independent experiments. Quantifications were performed by ArrayScan.
Atg16L1-FIP200 interacting domains.Figure 5.(A) Mapping of the Atg16L1-binding site on FIP200. Yeast two-hybrid mapping experiments showing that Atg16L1 binds to both the N-terminal and C-terminal coiled-coil regions of FIP200. (B) Mapping of the FIP200-binding site within Atg16L1. Yeast two-hybrid mapping experiments showing that the 1-840 fragment of FIP200 binds to the C-terminal 247-607 region, while the 1276-1591 fragment of FIP200 binds to the N-terminal 1-264 region of Atg16L1. (C and D) Additional yeast two-hybrid mapping experiments showing that the binding site of the FIP200 1276-1591 fragment maps to residues 239-246 of Atg16L1. White lines divide images derived from the same plate, and red lines divide images from different plates. (E) The N-terminal 1-249 fragment of Atg16L1 is sufficient to interact with FIP200. Full-length (lanes 1 and 2) or the N-terminal 1-249 fragments (lanes 3 and 4) of Myc-Atg16L1 coprecipitations with empty vector controls (lanes 1 and 3) or OSF-FIP200 1276-1591 (lanes 2 and 4). (F) Co-precipitation experiments confirming that OSF-FIP200 1276-1591 coprecipitates with wild-type (WT) Myc-Atg16L1 (1-249) fragments, but not with analogous fragments containing a mutant FIP200-binding site (Myc-Atg16L1 ΔWD 239-242A).
Figure 4A LincGET interact with hnRNP U, FUBP1, and ILF2in vitro. Silver staining of SDS-PAGE gel following RNA pull-down assay shows the proteins bound to LincGET (right lane) and antisense LincGET (α-LincGET, middle lane). Only one pull-down assay for mass spectrometry analysis was performed with 6,367 early 4-cell embryos. Three specific bands in the right lane (arrow) were analyzed through mass spectrometry and confirmed as hnRNP U, FUBP1, and ILF2. B. Mass spectrometry results of hnRNP U and ILF2 were confirmed by western blot following RNA pull-down assay (pulldown-WB). α-, anti-. For each pulldown-WB assay, about 1,500 early 4-cell embryos were used and three experimental replicates were performed. C. Expression pattern of Srsf1 in preimplantation mouse embryos measured by SG-qPCR. 1C, zygote stage. 2C, 2-cell stage. 4C, 4-cell stage. 8C, 8-cell stage. 16C, 16-cell stage. 32C, 32-cell stage. BL, blastocyst stage. The error bars represent s.e.m. About 50 embryos of each stage were used and three experimental replicates were performed. D. Co-IP results in early 4-cell embryos using anti-SRSF1 and in mouse ESCs using anti-SRSF1 or anti-HA (for HA-labeled MS2 coat protein). The results show that LincGET form an RNA-protein complex with hnRNP U, FUBP1, ILF2, and SRSF1. Moreover, hnRNP U, FUBP1, ILF2, and SRSF1 can form protein complex without LincGET. For each co-IP assay, about 2,500 early 4-cell embryos or 1 106 mouse ESCs were used and three experimental replicates were performed. E4C, early 4-cell embryos, mES, mouse ESCs, LincGET-, LincGET elimination, LincGET+, LincGET overexpression. E. Model showing that LincGET acts as a transcription factor and RNA alternative splicing factor through binding to hnRNP U, FUBP1, and ILF2.
Figure 3A. MDCK cells were transfected with siControl or the siRNAs indicated, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated. B. The intensity of the bands was measured in three independent experiments. C. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. D. Quantification of the NTA data obtained in five independent experiments.
Figure 5A Functional annotation charts (Benjamini, p<0.05) using using DAVID (GOTERM_Cellular Component) perfomed on the 82 significantly down-regulated genes by CSP-TTK21 treatment in tauopathic mice (TAU MOL vs. TAU VEH), showing a significant association with neuronal terms. Significance is indicated as -log10(Benjamini p_value). B Heatmap representing expression z-score of the 82 significantly down-regulated genes in the TAU MOL vs. TAU VEH comparison, for all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson"s dis C RNA-seq data showing expression of several genes belonging to « synapse » annotation (DAVID): Grik4, Homer3 and Sv2b, which are significantly down-regulated by CSPTTK21 treatment in THY-Tau22 mice. Adjusted p values corresponding to p<0.05 are indicated by * for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH D Venn diagram showing that a significant number of genes down-regulated by CSP-TTK21 treatment overlapped with genes up-regulated in the pathology (p=2.1.10-72. Khi2 test), as well as with some genes down-regulated by learning in WT (p=2.3.10-3, Khi2 test) Functional annotation charts using DAVID (GOTERM_Cellular Component) performed on the differentially regulated genes in Learning in the WT mice condition (WT VEH vs. WT VEH_HC). The down-regulated genes by learning (E) are associated with a neuronal terms, and up-regulated ones (F), with transcriptional and translational functions. Significance is indicated as -log10(Benjamini p_value)
Fig.Figure 4(A) Schematic representation of the KB-AT-23 expression cassettes used to test the secretion signal peptides SSP1, SSP2, SSP3, SSP4 and SSP5. hAAT: human alpha-1 antitrypsin promoter; His: N-terminal 6X-Histidine tag; HA: C-terminal tag derived from the human influenza hemagglutinin; BGHpA: bovine growth hormone polyadenylation signal. (B) Western blot analysis of KB-AT-23 protein secretion in conditioned media 72 hours post-transfection of HuH7 cells. In the lower panel the western blot quantification via densitometry analysis is depicted. Data are presented as mean ±SD, and were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons. ****p<0.001. (C) HuH7-celles were collected 72 hours post-transfection. Presented is the Western blot analysis of lysed cells for the presence of KB-AT-23. Cntr- refers to non-transfected cells, while Cntr+ refers to purified recombinant KB-AT-23.
β-catenin inhibits translation.Figure 5Primary VSMCs (left panel), A10 (middle panel), and HEK 293T cells (right panel) were transfected with siRNAs targeting ctnnb1 (β-catenin) or scrambled RNA. After 36 hr, the cells were pulsed with 0.5 µM puromycin for 15 min and then harvested. The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody. β-catenin blots indicated efficacy of siRNA and actin was used as a loading control. The average puromycin/actin ratio (n=3) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation. A one-way ANOVA and Tukey post-hoc test was performed, with p-values indicated above the error bars A10 cells were transfected with Flag-FMRP then treated with either cycloheximide or its solvent (DMSO) for 4 hr prior to fixation. β-catenin (green), Flag (FMRP, red) were visualized by immunofluorescence. Nucleus (blue) was identified by DNA staining with Hoechst 33342. Arrows indicate a loss of β-catenin signal from the perinuclear region that was seen in the control following cycloheximide treatment Schematic depiction of rapamycin mediated inhibition of cap-dependent translation A10 cells were transfected with Flag-FMRP as above and then pre-treated with rapamycin or solvent (DMSO) for 1 hr in serum-free media. Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β-catenin antibody. Non-programmed rabbit IgG was used as a control. Existence of Flag-FMRP in the immunocomplex was detected by Flag antibody and β-catenin by GFP antibody A10 cells were transfected with Flag-FMRP followed by rapamycin treatment similar to (D). Cells were fixed and stained for β-catenin (green), Flag (FMRP, red), and nucleus (blue). A loss of β-catenin signal from the perinuclear region that was seen in the control is indicated by the arrows.
Fig.Figure 1(A, B) qRT-PCR analysis of IL6 expression upon indicated stimulations in presence or absence of NOD1 (A) or NOD2 (B). HeLa inducible NOD1 cells (A) or NOD2 cells (B) were induced in absence or presence of doxycycline overnight and afterwards stimulated with various stimuli for 4 h. (C, D) ELISA analysis of IL6 production in supernatants of HeLa NOD1 cells (C) or NOD2 cells (D) upon different stimulations. HeLa NOD1 cells (C) or NOD2 cells (D) were induced in absence or presence of doxycycline overnight, stimulated with various stimuli for 20 h and afterwards supernatants were collected for ELISA. (E, F) Samples mean value of Eigengene analysis of TNFα signaling via NF-κB pathway (E) and inflammatory response pathway (F) of HeLa NOD1 or NOD2 cells upon indicated stimulations.
Figure 3Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences. A. ssG4: G4 flanked by single-stranded DNA; ssHP: hairpin flanked by single-stranded DNA; ss: single-stranded DNA Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences. B. Binding affinity of Timeless-Tipin for a range of G4 DNA sequences Single-stranded (ss20: 5′-6FAM-ATAAGAGTGGTTAGAGTGTA) and double-stranded (ds20: ss20 annealed to complementary sequence) DNA were also tested as controls.