EMBO/sd-character-level-ner huggingface dataset:
Overexpression of caldendrin leads to an increase of stable SER and the spine apparatus in dendritic spines of rat primary hippocampal neurons.Figure 5A Example confocal images from 10 min time-lapse imaging of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons (DIV17) expressing an ER-marker (ER-tDimer). Yellow arrows show dendritic protrusions with a stably inserted ER (>10 min). Blue arrows indicate protrusions with a transient ER entry. Scale bar = 5 µm. B Quantification of dynamic SER presence in protrusions of control (GFP) or caldendrin (cald-GFP) transfected neurons. In protrusions of cald-GFP neurons, the percentage of stably localized ER (> 10 min) is increased, while transient SER visits are decreased compared to the control. (mean ± SEM, 2-way ANOVA, * p=0.0386 for interaction, n GFP = 9, n cald-GFP = 10; 2 experiments) C Representative confocal images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons expressing an ER-marker (ER-tDimer). To pre-load overexpressed caldendrin with Ca2+, neurons were stimulated with 50 µM bicuculline for 5 min 8 hours prior to fixation and stained with a homer1-antibody to visualize synapse-containing spines. Arrows indicate spines that contain ER. Scale bar = 5 µm. D - F Quantification of protrusion- and spine density and the number of SER-positive spines as shown in C. (mean ± SEM, n GFP ctrl = 24 cells, n Cald-GFP = 25 cells; 3 experiments). (D) The density of dendritic protrusions (spines and filopodia) is increased in cald-GFP overexpressing neurons compared to the control. (* p=0.0119, unpaired t-test). (E) The density of homer1-positive dendritic spines is increased in cald-GFP overexpressing neurons compared to the control. (** p=0.0049, unpaired t-test). (F) The percentage of ER-containing dendritic spines is increased in cald-GFP overexpressing neurons compared to the control. (* p=0.0179, unpaired t-test) G G Representative confocal images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons pre-treated with bicuculline as in C and stained against homer1 and the spine apparatus-marker synaptopodin. Yellow arrows indicate synaptopodin clusters localized inside the dendritic shaft, blue arrows indicate spine-localized synaptopodin. Scale bar = 5 µm. H - J Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in G. (mean ± SEM, n GFP ctrl = 21 cells, n cald-GFP = 30 cells; 2 experiments). (H) Spine density is increased in cald-GFP overexpressing neurons compared to the control (in line with 5e). (** p = 0.0024, unpaired t-test). (I) The total number of synaptopodin clusters is not affected by caldendrin overexpression. (n.s., unpaired t-test). (J) The percentage of synapotpodin-positive spines is increased in cald-GFP overexpressing neurons compared to the control. (**** p < 0.0001, unpaired t-test).
Figure 4A-C Immunoblot analysis with anti-MYC antibody or anti-GFP antibody showing the upshift of ARR1, ARR10, and ARR12 in a phos-tag gel. The phosphorylated ARR1, ARR10, and ARR12 were abolished after treatment with calf intestinal alkaline phosphatase (CIAP). Seven-day-old seedlings of 35S:ARR1:MYC (A), 35S:ARR10:YFP (B), and 35S:ARR12:YFP (C) in Col and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points. Total proteins were extracted and subjected to immunoblot analysis. ARR1, ARR10, and ARR12 proteins separated on SDS-PAGE gel without phos-tag were detected as loading controls.
Widespread expression variability in Arabidopsis seedlings through the day and night A.Figure 1A. Experimental setup to identify transcriptional variability between seedlings during the day and night. RNA-seq was performed on individual seedlings, for a total of 14 seedlings at each time-point. Seedlings were harvested at 12 time-points, every 2 hours across a 24 hour period. Seedlings of different colors represent different transcriptional states, and thus inter-individual expression variability we aim to identify. B. Identification of highly variable genes (HVGs) for the time-point ZT2. The trend for the global relation (red line) between CV2 (variance/mean2) and mean expression is defined using all genes, minus small and lowly expressed genes (see material and methods for more detail) and used to identify HVG (blue). For each gene, a corrected CV2 is calculated: log2(CV2 /trend). C. Expression profiles (top) in the 14 seedlings over a 24 hour time course (with 12 time-points) for a non-variable gene (left), a highly variable gene (middle) and a gene with the level of variability changing across the 24 hour (right). Each dot is the mean normalised expression level for a single seedling. Variability profiles (bottom) of the log2(CV2 /trend) for the same genes are also shown. Blue stars indicate time-points for which the gene is identified as being highly variable.
Figure 4A. Co-purification experiments using His6-TssA1 and untagged TssB1C1. Western blot analysis of the Ni-NTA outputs using antibodies directed against the His6-tag (upper panel) and TssB1 (lower panel). I, Input; FT, Flow through; W, Wash and E, Elution. B. SEC elution fraction of the co-purified His6-TssA1 and TssB1C1. SDS-PAGE showing the presence of the three proteins His6-TssA1 (A1), TssB1 (B1) and TssC1 (C1). Molecular weight markers (kDa) are indicated on the left. C. Representative micrographs of the immunogold-labelled complex His6-TssA1/TssB1C1. The presence of gold particles is indicated with white arrows. Two close-up views of TssB1C1 sheath displaying gold particles at one extremity are shown (inset panels). Scale bars are 2,000 Å and 500 Å for images 1 and 2-6, respectively. D. BTH experiment showing interaction between TssA1 (A1) and Hcp1. A graphical representation of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.
Fig.Figure 1A. Infection by a L. pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification. Raw264.7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h. Saponin soluble proteins resolved by SDS-PAGE were probed with a UBE2N-specific antibody (top panel). The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins. Note that MavC but not MvcA is detectable in cells infected with wild type bacteria (middle panels labeled as "translocation"). Tubulin was probed as a loading control. Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin insoluble fraction (lower panels labeled as "expression"). The bacterial metabolism enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control. Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium (the middle panel of the lower portion). B. Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian cells. HEK293T cells were transfected with combinations of empty vector and plasmid directing the expression of MavC or MvcA. 16 h after transfection, modification of endogenous UBE2N was detected by immunoblotting (upper level). The canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis (Ypt), which cannot modify UBE2N, was included as a control. Note that coexpression of MvcA led to accumulation of unmodified UBE2N (4th lane) in samples that expressed MavC. The expression of MavC MvcA and Cif was detected using a Flag-specific antibody (lower panel). Tubulin was probed as a loading control (lower panel). C. MvcA interferes with MavC-induced UBE2N ubiquitination. Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by Coomassie Brilliant blue (CBB) staining. Note that MvcA (inclusion of MvcA with MavC, 7th lane. Compare to MavC alone in 3rd lane) but not the MvcAC83A mutant (8th lane) abolished MavC-induced UBE2N modification. D. Dose-dependent modification of UBE2N by MavC and MvcA. A series of reactions containing MavC and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min. Samples resolved by SDS-PAGE were detected by CBB staining.
Figure 3(A) FP titrations showing the binding of wild-type (WT) CaM to PSD-95 (1-21) peptides, either WT (blue), Q15A (salmon), E17R (black), T19E (green), or T19K (yellow). The solid lines represent optimal theoretical curves calculated using a one-site binding model described in Methods. The calculated dissociation constants in each case are given in Table 1. (B) FP titrations showing the binding of R126E CaM to PSD-95 peptides, either WT (blue) or E17R (black). (C) FP titrations showing the binding of K115E CaM to PSD-95 peptides T19K (yellow) or T19E (green).
Interactions of Okp1 or Ame1 with Nkp1-Nkp2.Figure 5A,B) Overlayed chromatograms showing absorbance at 280 nm from SEC of Ctf19-Mcm21-Okp1 variants without Okp1 segment 3 (A) or with Okp1 segment 3 (B); Ctf19-Mcm21-Okp1 variants: CMO, Nkp1-Nkp2: NN, Ctf19-Mcm21-Okp1 variants combined with Nkp1-Nkp2: CMO-NN; and images of SDS-PAGE gels with equivalent elution fractions from five SEC experiments; L: loaded on column. The elution-volume range that we analysed fractions of on SDS-PAGE is indicated below the chromatograms. C) Representative microscopy-images of living haploid S. cerevisiae cells with Okp1_fl (integrated full-length Okp1) or our Okp1 version that lacks the coding region for segment 3-Okp1_nnΔ, with Nkp1-GFP or Nkp2-GFP. Fluorescence images are maximum intensity projected, and we show those from Okp1_fl or Okp1_nnΔ with the same fluorophor-fusion kinetochore protein on the same intensity scale; scale bar: 2 μm. D,E) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Nkp1 (D) each in Nkp1-Nkp2 or COMA-Nkp1-Nkp2. Nkp1 has an N-terminal SNA residual. Plots for full time courses are in Appendix Fig S5A,B. D,E) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Nkp2 (E), each in Nkp1-Nkp2 or COMA-Nkp1-Nkp2. Nkp1 has an N-terminal SNA residual. Plots for full time courses are in Appendix Fig S5A,B.
BACE1 is modified with bisecting GlcNAc in vivo.Figure 1Bisecting GlcNAc modification by GnT-III. BACE1 or APP was immunoprecipitated from mouse brains and blotted with E4-PHA lectin (lower) or anti-BACE1 or APP antibodies (upper). hAPP indicates the APP23 transgenic mouse model for AD. Proteins from mouse brain membrane fractions were treated with or without PNGase F and then immunoblotted for BACE1 or for syntaxin 6 (loading control). LC-MS base peak chromatogram of desialo-alditol N-glycans derived from mouse brain BACE1. To simplify the results, N-glycans were chemically desialylated before LC-MS analysis. BACE1-specific glycans, judged by comparison with N-glycan structures from anti-BACE1 IgG (shown in Supplementary Fig S2C), are highlighted by red squares. Asterisks indicate glycans demonstrated by MS/MS analysis to contain a bisecting GlcNAc structure. Numbers in parentheses indicate the charge state.
Figure 4A. Replication fork speeds in U2OS or U2OS/GFP-RADX cells transfected with control (−) or RADX(#6) siRNA targeting the 3'UTR and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition). B. Fork symmetry in cells treated as in (A) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition). C. Proportion of stalled forks (CldU-only tracks) among DNA fibers in (A) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments). D. Proportion of new origins (IdU-only tracks) among DNA fibers in (A) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments). E. Parental U2OS and derivative lines stably expressing GFP-RADX alleles or all three RPA isoforms at near-endogenous levels (U2OS/Super-RPA) (Fig EV4A) were transfected with control (−) or RADX siRNA. Replication fork speeds were determined as in (A) (bars, mean; n=400 fibers, pooled from two independent experiments, per condition). F. Replication fork speeds in HCT116 cells with indicated genotypes transfected with control (−), RPA1 (0.2 nM concentration [17]) or RAD51 siRNAs (Fig EV4B) and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition). G. Fork symmetry among DNA fibers in (F) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition). H. New origin firing among DNA fibers in (F) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).
Figure 1(A) Molecular structure of ND-011992. (B) Effect of ND-011992 on the intracellular ATP level in M. bovis BCG. The bacteria were treated with DMSO (blue triangle), 100 nM Q203 alone (green triangle), 20 μM ND-011992 alone (brown square), and a dose-range of ND-011992 in the presence of Q203 (red squares) for 15 hours before quantification of intracellular ATP levels. (C) Oxygen consumption assay in M. bovis BCG using methylene blue as an oxygen sensor. (D) Oxygen consumption assay in M. bovis BCG using the Seahorse XFe96 Extracellular flux analyser. 12 μM ND-011992 was injected alone (green triangles), 100 nM Q203 (blue circles), or in combination with Q203 (red squares). For each condition, OCR readings were normalised to the last basal OCR reading before drug injection. OCR: Oxygen Consumption Rate. The dotted lines indicate the timepoints of drug injection. (E) Dose-dependent inhibition of M. bovis BCG OCR by ND-011992 (in the presence of 100 nM Q203) measured on a Seahorse XFe96 analyser platform. For each condition, OCR readings were normalised to the last basal OCR reading before drug injection. (F) Effect of ND-011992 on the OCR of M. smegmatis IMVs using the Oroboros O2k fluorespirometer. IMVs OCR from the parental strain (green triangles), ∆cydAB knockout (blue circles), and ∆cydAB complemented with M. tuberculosis CydABDC+ (red squares) energized with NADH were determined. Q203 was used at 1 μM. 100% OCR was defined as the OCR of the untreated samples for each strain.
Figure 4A. NLK induces YAP Ser 128 phosphorylation. Flag-YAP WT or S128A mutant was co-transfected with or without Myc-NLK. Phosphorylation was determined by Western blot using YAP S128 phosphospecific antibody. B. NLK phosphorylates YAP Ser 128 in vitro. NLK-WT or NLK-KN was transfected into HEK293A cells and immunoprecipitated. An in vitro NLK kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP Ser 128 was determined by immunoblotting with phospho-YAP (S128) antibody. C. Osmotic stress induces YAP phosphorylation at Ser 128. In the left panel, HEK293A cells were treated with sorbitol and endogenous YAP was immunoprecipitated. In the right panel, Flag-YAP was transfected into HEK293A cells. Flag-YAP was immunoprecipitated, YAP Ser 128 phosphorylation was detected by a phosphospecific antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3). D. NLK deficiency reduces YAP S128 phosphorylation. Two CRISPR/CAS9 gRNA plasmids targeting different sites of NLK were transfected into HEK293A. WT cells and two pools of NLK CRISPR/CAS9 transfected cells were treated with 0.2 M sorbitol for the indicated time points. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 4). E. The S128D phosphomimetic mutant abolishes YAP interaction with 14-3-3. Flag-YAP WT and mutant constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs. 14-3-3 was immunoprecipitated with Myc antibody and the associated YAP was detected with Flag antibody. F. YAP Ser 128 phosphorylation is required for disruption of YAP-14-3-3 interaction by osmotic stress. Flag-YAP WT or Flag-YAP S128A constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs, and treated with 0.2 M sorbitol for the indicated time points or refreshed with serum containing medium for 1 hr. Refreshing medium served as a control to disrupt YAP and 14-3-3 binding.14-3-3 was immunoprecipitated with Myc antibody, and the associated YAP was detected with Flag antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).
Figure 7A) Relative expression of α-tubulin and β1-tubulin in platelets from P1 and P3 (homozygous p.P160L), P4 (heterozygous p.Y106X), and P6 and P7 (heterozygous c.35delG) was quantified by Western blotting using specific antibodies against α-tubulin or β1-tubulin. With both mutations, α-tubulin expression was normal but β1-tubulin expression was significantly decreased. The β1-tubulin antibody recognizes C-terminal domain B) αIIbβ3 integrin activation was evaluated by flow cytometry (% of positive platelets and mean fluorescence intensity) in whole blood from P1 and P3 (homozygous p.P160L) and from P6 and P7 (heterozygous c.35delG); P4 could not be studied. While no αIIbβ3 integrin activations were detected in P6 and P7 (family 3), P1 and P3 (family 1) exhibited a significant increase of αIIbβ3 activation, indicating basal platelet activation C) Aggregation of washed platelets induced by ADP (5 or 10 μM) or collagen (0.6, 0.8 or 2 μg/mL) was evaluated in at least one member of each family. Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls, suggesting that TUBB1 affected by homozygous p.P160L and heterozygous p.Y106X mutations affected platelet function. Platelet aggregation to both agonists was normal in both patients from family 3 (heterozygous c.35delG). Family F1, n=3 independent experiments; Family F2, n=1 experiment; Family F3, n=2 independent experiments. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, nd: statistical significance not determined because
Figure 4Double immunolabeling of PDS5A (green, A-E) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages. Double immunolabeling of PDS5B (green, F-J) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages. K-M Preparations from the same mice were immunolabeled with the testis-specific histone variant H1t (green) and SYCP3 (red). N Scatter dot-plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype. Bars indicate mean and standard error. Statistical significance was assessed using an unpaired two-tailed T-test function (****, p < 0.0001).
Figure 2A. Elution profiles and SDS-PAGE for SEC runs on the indicated column of the Cyclin B1:CDK1 complex (blue profile), MBP-MAD1:MAD2 (red), and their combination (green). Note that the same Cyclin B1:CDK1 elution profile and SDS-PAGE is also displayed as reference in panels D and F to improve clarity. For the same reason, the MBP-MAD1:MAD2 elution profile and SDS-PAGE is also displayed in panels 2A-C. B. Elution profiles and SDS-PAGE for SEC runs on the indicated column of CDK1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). C. Elution profiles and SDS-PAGE for SEC runs on the indicated column of Cyclin B1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). D. Elution profiles and SDS-PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD1∆93:MAD2 (green), and their combination (red). F. Elution profiles and SDS-PAGE for SEC runs of Cyclin B1:CDK1 (blue), MBP-MAD13EK:MAD2 (E52K, E53K, and E56K mutations; red), and their combination (green). In A-D, individual potential binding partners were combined at a concentration of 5 µM. G. Alignment of the N-terminal region of Cyclin B1 that contains the MAD1 binding region. Numbering refers to the human MAD1 sequence. *conserved, negatively charged residues in MAD1 (E52K, E53K, E56K) required for MAD1:Cyclin B1 interaction.
Figure 3. (A) Metagene analysis of NET-seq signal at all DNC loci (n=1517). The genomic intervals were centered at the transcription start site (TSS) of either protein-coding gene (left panel) or DNC transcript (right panel) . (B) NET-seq data at the YPL172C locus. The signal represents NET-seq reads showing nascent transcription at the genomic position of the divergent non-coding strand (black) and the coding strand (grey). The bar graph depicts NET-seq reads as FPKM values for the coding and DNC transcript in the strains . (C) NET-seq data at the YDR261C locus with a novel DNC transcript. Annotations as in (B)
Pex19 overproduction and PEX19 or PEX25 deletion.Figure 5.(A and B) WB analysis (A) and protein quantification (B) in WT (lane 1; blue), pex3 atg1 (lane 2; red), and pex3 atg1-PAOXPEX19 (lane 3; green) cells grown for 6 h on MM-M/G. The protein levels in WT were set to 100%. (C and D) FM images (C) and quantification (D) of Pmp47-mGFP and Pex10-mGFP in pex3 atg1 (1; red) and pex3 atg1-PAOXPex19 (2; green) cells grown for 6 h on MM-M/G. Control cells in D did not produce mGFP. Significance indications: n.s., P < 0.10; *, 0.10 > P > 0.05; **, 0.05 > P > 0.01; ***, P < 0.01. Error bars indicate SD. (E and F) iEM analysis of pex19 (E) and pex19 atg1 (F) cells using α-Pex14 antibodies. (G) EM analysis of KMnO4-fixed pex3 atg1 pex25 cells grown for 16 h on MM-M/G-showing membrane vesicles (arrows). (H) FM image of pex3 atg1 pex25 cells producing Pex14-mGFP. CW, cell wall; M, mitochondrion; N, nucleus; V, vacuole.
SV2 is specifically reduced at neuromuscular junctions of symptomatic transgenic GA149-CFP mice Muscle sections (30μm) were cut from snap frozen tissue of 20-month old GA149 mice (Tgx GA149) or wild type controls.Figure 10Muscle sections (30μm) were cut from snap frozen tissue of 20-month old GA149 mice (Tgx GA149) or wild type controls. Immunohistochemical staining was performed to assess synaptic protein levels in proximity to NMJs. Sections were immunostained for SV2 or Synaptophysin, and counterstained with neurofilament. A) Left Panels: Representative images show SV2 levels (green) in proximity to α-bungarotoxin positive NMJs (red) in wild type animals (top) versus GA149 animals (bottom). Right Panels: Representative images show SV2 levels (green) overlapping with neurofilament (blue) at synaptic terminals approaching muscle in wild type animals (top) versus GA149 animals (bottom). B-C) Quantification of SV2 (B) or Synaptophysin (C) colocalized with neurofilament and normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in GA149 animals compared with wild types, while synaptophysin remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 10 non-overlapping regions of muscle from each of 5 animals per group were evaluated.