EMBO/sd-character-level-ner huggingface dataset:
Interaction between HsAtg4B and LC3.Figure 2(A) Interaction between HsAtg4B and the ubiquitin core of LC3. HsAtg4B is coloured salmon red, and LC3 is coloured green. The side chains of the residues involved in the HsAtg4B-LC3 interaction are shown as stick models. Possible hydrophilic interactions are shown as broken lines. (B) Interaction between HsAtg4B and the C‐terminal tail of LC3. Trp142 and the regulatory loop of HsAtg4B are coloured blue, whereas the remainder is coloured as in (A). As the side chain of Cys74 has two conformations, both conformations are shown. Possible hydrophilic interactions are shown as broken lines. (C) In vitro proteolysis assay. Left, LC3 mutants fused to GST were incubated with HsAtg4B. Right, LC3-GST was incubated with HsAtg4B mutants. Procedures are described in detail in Materials and methods.
Figure 4Relative mRNA expression of ATGL, PGC1α, PLIN5, and HSL in C2C12 cells transfected with ATGL (n=5). Relative mRNA expression and western blot analysis of ATGL in C2C12 cells transfected with Ant-183/96 and siRNA of ATGL (siATGL) as indicated (n=3 per group for RT-PCR analysis). Relative mRNA expression (C) and western blot analysis (D) of PGC1α, PLIN5, and HSL in C2C12 cells transfected with Ant-183/96 and siATGL as indicated (n=3 per group for RT-PCR analysis). Relative mRNA expression of ATGL in C2C12 cells transfected with Ago-183/96 and FoxO1 as indicated (n=5 per group for RT-PCR analysis). western blot analysis of FoxO1 and ATGL in C2C12 cells transfected with Ago-183/96 and FoxO1 as indicated (n=5 per group for RT-PCR analysis). Relative mRNA expression (G) and western blot analysis (H) of PGC1α, PLIN5, and HSL in C2C12 cells transfected with Ago-183/96 and FoxO1 as indicated (n=5 per group for RT-PCR analysis). Relative mRNA expression of PGC1α, PLIN5, and HSL in C2C12 cells transfected with Ant-183/96 and siRNA of PPARδ (siPPARδ).
Fig.Figure 4(A) The in vitro import of Mrp17-DHFRmut is sensitive to the elimination of outer mitochondrial membrane proteins by trypsinization (left), more so than the model import substrate Atp1 (right). Mitochondria were incubated with the indicated concentrations of trypsin and the import assay was performed as described in the legend for Fig. 2. Proteinase K (PK) was not added to the sample without mitochondria. (B) In vitro translocation of Mrp17-DHFRmut into mitochondria isolated from WT and ∆tom20 yeast showing reduced translocation in ∆tom20 background. (C) Quantification of the experiment in panel (B) repeated 3 times, whiskers correspond to minimum and maximum values. (D) Expression of Mrp17-GFP in ∆tom20 yeast shows mislocalization to the nucleus compared to WT cells (Full micrographs and WT control shown in Appendix Figure S6A). Scale bar is 10 µm. (E) Import of Mrp17-DHFRmut into purified ∆tom22 mitochondria, control import into WT mitochondria is shown on Appendix Figure S6B (F) Substitution of both Tom20-binding motifs (TBM) with alanines (TBM1,2mut) abolishes Mrp17 mitochondrial import capacity while substituting of a single motif (TBM1mut, TBM2mut) reduces import. (G),(H) Import of Mrp17-DHFRmut and control proteins Atp1 and Aac1 into WT, tim17-ts (G) and tim22-ts (H) mitochondria. Import was performed at indicated temperatures, proteinase K was added to all samples except the loading control in the first lane of each autoradiograph (20% of protein amount used for each import reaction). The full autoradiograph for Aac1 with molecular weight markers is shown in Appendix Figure S6D.
Overview of proteins of the yeast kinetochore.Figure 1The Cse4-containing nucleosome is depicted with the extended N-terminus, which carries R37Me and K49Ac. COMA subunits are shown in colour.
COPII and PI3K-dependent generation of small vesicles from ERGIC for LC3 lipidation.Figure 2.(A) Diagram showing the cell-free system to generate small vesicles for LC3 lipidation. Briefly, a medium-speed membrane pellet (20K ×g, 20KP) from Atg5 KO MEFs was harvested and incubated with cytosol from Atg5 KO MEFs, GTP and an ATP regeneration system (ATPR), and in the absence or presence of PI3K inhibitors (Figure 2B) or the PI3P blocker FYVE domain protein (Figure 2-figure supplement 1). The slowly-sedimenting membranes generated were separated from the donor membrane by a medium speed centrifugation (20K ×g) and collected by high-speed sedimentation (100K ×g) of the supernatant fraction (20KS). A lipidation assay with HEK293T cytosol was performed to analyze the competency of the small membranes (100KP) to induce LC3 lipidation. (B) A small vesicle generation assay as above was performed with the indicated conditions. Slowly-sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction. 3-MA, 5 mM; Wortmannin, 20 nM; S, starved Atg5 KO MEF cytosol; N, untreated Atg5 KO MEF cytosol; −/AP, in the absence of ATP regeneration and GTP and in the presence of apyrase (AP). (C) Diagram showing the fractionation procedure and fractions collected for the small vesicle generation assay shown in (D). Briefly, Atg5 KO MEF membranes were subjected to differential centrifugation to collect 3K ×g and 25K ×g pellet fractions. Sucrose gradient ultracentrifugation was performed to separate the L and P fraction after which the L was further separated by OptiPrep gradient ultracentrifugtion. Ten fractions from top to bottom were collected and combined to four as indicated (F1-4). (D) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K, P and F1-4 fractions (upper four panels: Small vesicle). SDS-PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction (lower five panels: Fraction input). TFR, transferrin receptor (E) A small vesicle generation assay as shown in (A) was performed in the presence of indicated proteins or drugs. Slowly-sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST, H79G (Sar1A (H79G), 0.7 μM), wortmannin (20 nM), or BFA (brefeldin A, 0.5 μg/ml). (F) A small vesicle generation assay as shown in (A) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A (H79G) or 3-MA followed by a lipidation reaction. (G) Atg5 KO MEFs were starved for 30 min. Differential centrifugation as shown in Figure 1D was performed to collect the 25K ×g and 100K ×g pellet fractions. The LC3 lipidation was performed on these two fractions with indicated concentrations of SAR1 (H79G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).
Figure 2A. SHOXprotein representation and luciferase assay. The variants found in SHOX are indicated in red. Luciferase assays to test the impact of SHOX mutations on its transcriptional activity were performed on FGFR3 promoter in U2OS cells (n = 4). Experiments were performed in triplicates. pcDNA4-TO empty vector was used as control. R153L represents a mutation known to affect SHOXprotein activity and was used as positive control (Schneider et al., 2005). Homeodomain, DNA binding domain; Src Homology 3 domain; OAR, OtpAristalessRax domain. RLU, Relative Light Units. B. CYP26C1protein representation and luciferase assay. Variants found in CYP26C1 are indicated in green. Cignal-RARE system luciferase assays to test the impact of CYP26C1 variants on its RA degradation activity were performed in U2OS cells treated with 250 nM all-trans retinoic acid (ATRA) for 24 hours (n = 4). Experiments were performed in triplicates.pIRES2-EGFP empty vector was used as control. The residue C459 represents the Iron binding residue (Q6VOL0, UniProtKB) and was mutated to Ala and used as a positive control. TM, Transmembrane helix; P450, cytochrome p450 domain. RLU, Relative Light Units.
Model of the MYC-miR-15a-TSC1 axis controlling mTORC1 activity.Figure 6Hyperactivation of mTORC1 through loss of TSC1 function results in metabolic stress, ROS production and apoptosis. Under this condition, low dose of rapamycin restores mTORC1 levels that are compatible with cell viability.
Placensin secretion and promotion of trophoblastic cell cAMP production, PKA activity, MMP9 expression, and invasion.Figure 4A Left panel: Immunoblotting of placensin content in HTR-8/SVneo, but not BeWo, JAR, and JEG3, cells. Right panel: Time-dependent secretion of placensin by HTR-8/SVneo, but not JAR, cells monitored using an ELISA (biological replicates, n=4-5). B Knockdown of FBN2 in HTR-8/SVneo cells led to decreases in FBN2 transcripts and placensin secretion. Cells were treated with FBN2 siRNA (SiFBN2; 20 nM) or control siRNA (SiC; 20 nM) for 6h. Following media change, cells and conditioned media were collected at 48h after incubation. Placensin transcripts and proteins were determined by RT-PCR and ELISA (biological replicates, n=8), respectively. Lower panel: immunoblotting of secreted placensin by HTR-8/Svneo. C Placensin stimulation of cAMP production and PKA activities by HTR-8/SVneo cells. Cells were treated with placensin for 10 min. before cAMP measurement (biological replicates, n=5). For PKA activities, cells were treated with placensin (50 ng/ml) for 30 min. Treatment with forskolin (10 uM) served as positive controls. D Placensin stimulation of transcript levels for MMP9 but not MMP2(biological replicates, n=12). Cells were treated with placensin for 24h before RT-PCR analyses of transcript levels. E Placensin stimulation of MMP9, but not MMP2, activities without affecting cell proliferation. After 24h of treatment with placensin, media were collected for zymographic and densitometric analyses as well as determination of total cell numbers (biological replicates, n=4). F Placensin stimulation of HTR-8/SVneo cell invasion. After placensin treatment for 24h, cell invasiveness was determined using the Transwell assay (left panel: micrographs of cells; right panel: fold changes in number of migrated cells, biological replicates, n=4). Cells were incubated with mitomycin C to rule out effects on cell proliferation. Scale bars: 100μm. C: control.
Figure 3(A) Detection of SARS-CoV-2 in nasopharyngeal swabs and peripheral blood using the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup S.p.A., Turin, Italy). Three viral target genes, RdRp, N, and E, together with the housekeeping gene GAPDH were simultaneously amplified. Here the most sensitive target gene, N, is shown. Samples with Ct values > 40 were defined as negative. A dashed horizontal line indicates the cut-off. IL-6 values are shown in the same graph. Time of baricitinib treatment is highlighted (Patients A, B, and D for 10 days, Patient C for 12 days) with total time of evaluation on the X-axis. Fever and cough symptoms are indicated. The bars in grey indicate that cough has improved but has not resolved. (B) Levels of CD3+, CD4+, CD8+, B cells (CD19+) and NK cells (CD3-, CD16+, CD56+) were determined using the AQUIOS CL Flow Cytometry System (Beckman Coulter). (C) Total serum IgG, IgA, and IgM levels are shown for all patients.
Figure 6 A Unmodified ReNcell VM human neuronal progenitor cells were co-plated with comparable Ren-GFP or Ren-APPSL cells into 3-dimensional Matrigel matrices, and differentiated into neurons for 60 days. Cytosolic GFP is produced by both Ren-GFP and Ren-APPSL cells, and the latter also produce pathogenic human APP with the Swedish and London mutations, and secrete Aβ that becomes entrapped in the Matrige Following neuronal differentiation, 2P-FLIM was used to monitor NiMA in the ReNcell VM neurons, which were discriminated from Ren-GFP and Ren-APPSL neurons because they lack GFP. Note that NiMA was observed in ReNcell VM neurons when they were co-cultured with Ren-GFP neurons, but not when they were co-cultured with Ren-APPSL neurons. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test  B ReNcell VM cells were differentiated into neurons for 30 days in 2D cultures. Subsequent AβO exposure for 16 hours blocked NiMA, but expression of Flag-Raptor-Rheb15, which forces mTORC1 to lysosomes, prevented NiMA inhibition by AβOs. Each colored line refers to a single field of view containing 5-15 cells, and each identically colored pair of solid and dotted lines refers to the same field of view. Flag-Raptor -Rheb15 expression was confirmed by western blot­ting. Statistical analyses were performed using Student's two-tailed unpaired t-test. Western blots are representative of 3 independent assays
CatSper inhibitors abolish chemotaxis of sperm in an chemoattractant gradientDark‐field microscopy images of a sperm suspension before (top) and after (bottom) photorelease of a resact gradient (middle) in the absence (control; left) or presence of the CatSper inhibitor MDL12330A (10 μM; right).Figure 7A Dark‐field light microscopy images of a sperm suspension before (top) and after (bottom) photorelease of a resact gradient (middle) in the absence (control; left) or presence of the CatSper inhibitor MDL12330A (10 μM; right). MDL12330A abolishes resact‐induced sperm accumulation.B Relative change of the sperm dispersion in the field of view evoked by photorelease of resact (t = 0, flash) in the absence (control; blue) or presence of MDL12330A (red); a decrease in dispersion indicates sperm accumulation in the irradiated area (mean ± SD; n = 4).
The hinge region is critical for activation of GC‐G by cool temperatures A Representation of the domain structure from different C‐terminal fragments of GC‐G analyzed for coolness‐induced activity (amino acids of GC‐G encoded in each expression plasmid are indicated).Figure 3A Representation of the domain structure from different C‐terminal fragments of GC‐G analyzed for coolness‐induced activity (amino acids of GC‐G encoded in each expression plasmid are indicated). B, C Protein expression and coolness‐stimulated cGMP synthesis of the above shown divergent C‐terminal fragments of GC‐G with intact or ablated hinge (H) region. The expression plasmids encoding the H+CYC domain or a deletion construct (Δ1-Δ2) tagged with a FLAG epitope were transfected into HEK‐293T cells. Two days after transfection, cells were exposed to the ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured (C). Protein expression of each construct was confirmed by Western blot (WB) analysis with anti‐FLAG antibody (B). D A recombinant protein containing the H+CYC domain of GC‐G acquires coolness‐evoked activity. Diagram of recombinant GST.GC‐G‐CYC protein containing the H+CYC domain of GC‐G (amino acids 849-1,078) compared to the domain structure of full‐length GC‐G (upper panel). The purified GST.GC‐G‐CYC protein was exposed to the indicated temperatures for 20 min, and GST.GC‐G‐CYC cyclase activity was measured (lower panel; asterisks denote values significantly elevated compared to 37°C).
Figure 3Volcano plot of the BCL9 mitotic interactome by the SILAC proteomics approach. Red dashed line indicates a strict cut off by a normalised SILAC H/L ratio > 2. DAVID functional annotation analysis of enriched biological process (BP) by the potential mitotic BCL9 interactome (normalised SILAC H/L ratio > 1.5). An independent immunoprecipitation assay for validation of potential BCL9 interactors in both asynchronised and mitotic cells. The relative binding of CLTC with BCL9 is quantified and labelled on top of the CLTC blot. Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells, from G1S to G2M. P-H3S10, a mitotic marker indicating mitosis.
Figure 1(A) Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 and fused in all three possible frames with the GFP deleted of its ATG. (B) LC-MS/MS spectra of the N-terminal part of the GFP-immunoprecipitated and LysC digested polyGA protein expressed as in (A). (C, D) Immunoblotting against the GFP (C) or the HA tag (D) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 3, 20 or 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame. (E) Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected with either a wild type or mutant (AGG into CGG) construct containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GR frame. (F) Scheme of human C9ORF72 sense transcript with intron 1 retained. Expanded G4C2 repeats, CUG and AGG near-cognate initiation codons, polyGA and polyGR ORFs are indicated in red. C9ORF72 ORF is indicated in blue.
Identification of pharmacologically distinct substrate binding sites.Figure 4A Inhibition of substrate crosslinking by the GSIs identifies substrate residues binding in the active site. L, L-685,458; D, DAPT. B Preventing access to the active site by GSIs identifies substrate residues interacting with the exosites by their increased labeling intensity. C Summary of effects of L-685,458 on C99 binding to γ-secretase. Note that crosslinking of almost all residues of the K54-I60 region to PEN-2 was increased in the presence of L-685,458 indicating additional exosites in this subunit binding to the juxtamembrane region of the C99 intracellular domain. See also Figs EV1 and 2 and Discussion. D Model of substrate binding in the presence of GSIs showing substrate accumulation at exosites provided by NCT, PEN-2 and the PS1 NTF.
mEPSCs and [Ca2+]i in brain slices.Figure 6(a, b) example traces of mEPSCs recorded from wild-type (a) and Trpml1-/-(b) micebrain slices as detailed in Methods. The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale. (c) histogram distribution of the events at each mEPSC. The lower columns show the distribution of the higher amplitude mEPSCs. (d) the frequency of mEPSCs in each line. The results in (c, b) are the mean±s.e.m of 8 experiments each obtained from 4 mice of each line. (e) the mean±s.e.m of the Ca2+ signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild-type and Trpml1-/- slices, respectively.
Figure 1A, Clustal alignment analysis of Trnp1 orthologs. Highly conserved and similar residues are shown in black and grey, respectively. Asterisks show 100% conserved residues among all orthologs. Predicted disordered and structured regions are depicted with a red line and a black line, respectively. Cylinders represent alpha-helices and arrows represent turns. B, Analysis of the coiled-coil structure of Trnp1 using the prediction tool COILS. C, Immunostaining of endogenous Trnp1 and DAPI in P19 cells and E14 cortical cells at 1 day in vitro (div). D, Co-IP with anti-GFP and WB with anti-Trnp1 antibodies in lysates from P19 cells transfected with plasmids expressing Trnp1-GFP fusion protein and/or Trnp1-IRES-RFP for 24h. Mw (molecular weights): 55 kDa (black line); 35 kDa (blue line); 25 kDa (red line). Input: 0,1%; co-IP: all immunoprecipitated proteins. E, Representative phase contrast images of in vitro phase separation of the recombinant proteins YFP (upper row) or Trnp1 (lower row) at the indicated concentration in 50 mM Sorenson's buffer (pH 7,6) containing 150 mM salt and 2mM DTT with the crowding agent dextran or RNA when depicted.
Figure 4a-c, hippocampal neurons were infected with rAAVs expressing GFP (control) or Npas4 (Npas4 overexpression) on DIV4. On DIV11, RNA was harvested and subjected to microarray transcript analysis. a, overexpression of Npas4 leads to ≥ 1.5-fold upregulation of the levels of 1091 sequences and to a ≥ 1.5-fold downregulation of the levels of 1133 sequences, indicating that Npas4 regulates a widespread transcriptional response. b, gene ontology analysis (GO) for biological processes via the Panther tool for up- and downregulated genes from panel a). Shown is a selection of relevant processes. Upregulated genes are enriched for cell-signaling processes (i.e., TGF-beta signaling, hormone responses), behaviour (i.e., locomotion) and developmental processes (i.e., anatomical structure development). Downregulated genes are enriched for synapse regulation (i.e., regulation of synapse assembly), dendritic organization (i.e. dendrite morphogenesis) and cognitive processes (i.e., learning, cognition, locomotion). c, selection of genes from the Npas4 regulon with relevance to synaptic transmission and dendrite morphology, both of which are processes implicated in habitual learning. d, Npas4 overexpression leads to basal upregulation of genes encoding for proteins targeted to glutamatergic synapse function and Ca2+ channels in MSNs. MSNs were infected with rAAVs expressing mCherry-NLS control or Npas4 on DIV4 or DIV5 and expression of the indicated genes was analysed by qPCR on DIV11 or DIV12. Shown are mRNA levels normalized to mCherry-control. The dashed line represents normalized control levels. pCAG = CAG promoter, myc = myc-tag, flag = flag-tag. Mean + SEM; unpaired, two-tailed t-test for each gene, p*<0.05, p**<0.01, p***<0.001. n = 5 independent cultures as biological replicates. e, Npas4 controls activity-dependent upregulation of genes for glutamatergic synapse formation and Ca2+ channels. MSNs were infected with rAAVs expressing shRNA-scrambled (control) or shNpas4 (Npas4 knockdown) on DIV5. On DIV 12, cultures were stimulated with HiK (55 mM) for 1h or 6h and mRNA levels were analysed by qPCR. mRNA level = fold change in mRNA compared to shscrambled unstimulated control. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p***<0.001. n = 5 independent cultures as biological replicates.
Figure 4A, B, C The layer thickness and neuronal number within each layer was assessed using layer-specific markers in Kcc2-/-and Kcc2+/+ embryos at E18.5. Cux1 was used to label layers II-IV (A), Ctip2 to label layer V (B), and Tbr1 to label layer VI (C). Dashed white lines in the representative images in (A-C) indicate upper and lower layer boundaries. Sp, subplate. Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc (neuronal numbers) and one-way ANOVA with Holm-Sidak's post hoc (layer thickness). Data are presented as mean ± S.E.M., n = 11 embryos for both genotypes. Scale bar = 50 µm. D Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) in Kcc2-/- and Kcc2+/+ embryos electroporated in utero at E14.5 with a plasmid encoding EGFP (green). DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. E Neurons from the brain sections in (D) were quantified and the total number of EGFP+ neurons in the Kcc2-/- sections is presented as a percentage of pooled Kcc2+/+ and Kcc2+/- values. Statistical significance was determined using one-tailed Student's t test, *P < 0.05. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos. F Number of EGFP+ neurons in Kcc2-/- embryos migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective pooled data from Kcc2+/+ and Kcc2+/- embryos. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01 to Kcc2+/+ + Kcc2+/-. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos.