EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 1(A) Total RNA from mock infection or infected cells was depleted of rRNA and cDNA libraries were prepared. Libraries were then either indexed and sequenced directly for host transcripts or enriched using pathogen specific oligonucleotides bound to beads. After hybridization, enriched libraries were indexed, sequenced and reads assigned to host or pathogen genomes in silico. (B) Correlation between replicate mock infections. Path-seq reads were recovered from samples of macrophage RNA spiked with 0.1% MTB RNA. Scatter plot of log2 RPKM values is shown with Pearson correlation, P-value < 0.0001. Inset summarizes the mock infection replicates and their fraction of MTB reads (of all aligned reads) from Path-seq and standard RNA-seq methods. (C) Correlation between MTB RNA sequenced by RNA-seq (Illumina TruSeq library prep) and the same MTB RNA sample combined with macrophage RNA at 1:1000 ratio and processed using Path-seq method. Scatter plot of log2 RPKM values is shown with Pearson correlation, P-value < 0.0001.
HIV induces TFEB nuclear localization in human macrophages through TLR8.Fig 4(A) Macrophages were exposed to increasing concentrations of purified HIV for 24 h, harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (B) Macrophages were infected with HIV and at 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6. C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; green) and antibody to TFEB (red) at the indicated times post-infection. Co-localization of TFEB and DAPI staining is represented in the image by orange pixels. Scale bars indicate 20 μm. (D) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h, harvested, lysed fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (E) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (F) Macrophages transduced with shNS or shTLR8 from (E) were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were then harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
Figure 6Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) and NU-7441 was administered intraperitoneally (i.p.; 10 mg/kg/day), over the indicated periods of time. Vertical dashed line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 6 mice. Error bars represent SEM. P values were calculated at day 24 using an unpaired two-tailed t-test N.S. P> 0.5; **, P ≤ 0.01; ****, P ≤ 0.0001. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) over the indicated periods of time, and paclitaxel (PACLI) was administered intravenously (i.v.; 20 mg/kg/day) on indicated single days. Vertical dashed line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 6 mice. Error bars represent SEM. P values were calculated at day 24 using an unpaired two-tailed t-test ****, P ≤ 0.0001. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day), NU-7441 was administered intraperitoneally (i.p.; 10 mg/kg/day), over the indicated periods of time, and paclitaxel (PACLI) was administered intravenously (i.v.; 20 mg/kg/day) on indicated single days. Vertical dashed line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 6 mice. Error bars represent SEM. P values were calculated at day 36 or 38 using an unpaired two-tailed t-test ****, P ≤ 0.0001. Kaplan-Meier survival curve for mice treated as in (C), with each treatment group containing n = 6 mice. Statistical significance was assessed by Log-rank test (****P <0.0001).
Figure 1(A) Representative confocal images of muscle-6/7 NMJ synapses of abdominal hemisegments A2-A3 for the indicated genotypes labelled with anti-Synaptotagmin (green) and HRP (red) to reveal the synaptic vesicles and the neuronal membrane. Scale bar: 20 μm. (B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction. (n.s. = not significant; p<0.05 = *; p<0.001= ***; p<0.0001=**** Data information: In (B-D bars are labelled with the number of replicates. (E) Immunofluorescence analysis of adult brains for the indicated genotypes using anti-Fascilin II (FasII) antibody to visualize lobes of the MB. Pink dashed lines highlight the normal and fused β-lobes. Scale bar 50 μm. (F) Quantification of the penetrance of fusion phenotype in in the indicated genotypes. Data information: In F), bars are labelled with the number of replicates.
Figure 3(A) Schematic diagram of PLZF and truncated PLZFs. FL; Full-length, BTB; BTB (broad-complex, tramtrack, and bric à brac) domain. (B) In vitro binding assay using truncated PLZF. Thalidomide-dependent interaction between bls-CRBN and FLAG-GST-PLZF-full length (FL) or truncated FLAG-GST-PLZF was analysed in the presence of DMSO or 50 µM thalidomide using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, ****P < 0.0001 (C) Immunoblot analysis of AGIA-PLZF protein levels in FLAG-CRBN and PLZF-full length (FL) or truncated FLAG-GST-PLZF expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. (D) Schematic diagram of swapped PLZF mutants. (E) In vitro binding assay using swapped PLZF mutants was performed Data information: All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, ****P < 0.0001). (F) Amino acid sequences of ZNF1 and ZNF3 in PLZF. The white letters indicate critical Gly residue for the interaction between PLZF and CRBN with thalidomide or its derivatives. (G) In vitro binding assay using point mutants of PLZF was performed Data information: All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, ****P < 0.0001). (H) Immunoblot analysis of AGIA-PLZF protein levels in FLAG-CRBN and PLZF-WT, PLZF-ZNF1-GA, PLZF-ZNF3-GA, or PLZF-ZNF1,3-GA expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h.
Figure 1 (A) Time-course SUMO conjugation reaction in the presence or absence of ssDNA (virion ϕx174) using recombinant full-length Smc5-Nse2 complex. The substrate utilized was the C-terminal kleisin domain of Nse4 (cNse4). Reaction was run at 30°C and stopped at indicated minutes by adding SDS-loading buffer. Labels indicate the bands in the SDS-PAGE of the proteins in the reaction mixture (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2 and N-3S2, cNse4-3SUMO2)  (B) Left, SYPRO-stained and Western blot (anti-SUMO2) of the SUMO conjugation reaction by Smc5-Nse2 complex in the presence of ssDNA (virion ϕx174) and dsDNA (pET-DUET-1) at either 1 or 10 nM. Reactions were run at 30°C and stopped at 60 minutes by adding SDS-loading buffer. Right, bar diagram comparison of the relative SUMO conjugated cNse4 substrate in the presence of either ssDNA (virion ϕx174) or dsDNA (pET-DUET-1) at indicated concentrations. Straight line shows the basal cNse4-SUMO conjugation in the absence of DNA. Data values are mean ± s.e.m. and n=3 technical replicates  (C) Left, Western blot of the SUMO conjugation reaction in the presence of 50b oligonucleotide and the 5 kb virion ϕx174 at the indicated concentrations. Reactions were run at 30°C and stopped after 60 minutes by adding SDS-loading buffer. Right, bar diagram comparison of the relative SUMO conjugated cNse4 substrate in the presence of 25, 34 or 50 bases oligonucleotides at indicated concentrations. Straight line shows the basal cNse4-SUMO conjugation in the absence of DNA. Data values are mean ± s.e.m. and n=3 technical replicates. Bar diagrams calculation was generated using ImageJ software (Schneider et al, 2012)
EM reconstruction of endogenous holoElongator.Figure 1A. SDS-PAGE gel showing the purified holoElongator complex used as input for gel filtration. Protein bands marked with asterisks presumably correspond to different phosphorylation states of Elp1 that have been previously described [34]. B. Representative negative stain-EM field of the holoElongator complex. Particles in side and tilted views are highlighted. Scale bar, 50 nm. C. Reference-free class averages and back-projections of the holoElongator model. Scale bar, 20 nm. D. EM reconstruction of the holoElongator complex at 31 Å resolution.E. Fitting of the Elp456 crystal structure into the ring density of the holoElongator reconstruction.F. Fitting of the Elp1 CTD into the holoElongator reconstruction.
Figure 3A The schematics of the WT and the mutant (∆5′/∆3′) with deletion of the 5′ and 3′ terminal regions are shown as Figure 1A. The positions of the NEAT1 probes used in SRM (blue) and EM (orange) are shown. B SRM images of the paraspeckles in MG132-treated (5 μM for 6 h) HAP1 NEAT1 ∆5′/∆3′ cells detected by NEAT1_2k (green) and NEAT1_15k FISH probes (left, magenta) or NONO immunofluorescence (IF) (right, magenta). Scale bar, 500 nm. C Graph showing the proportion of paraspeckles with localization of the NEAT1 5′ (upper; WT: n = 41, ∆5′/∆3′ kb: n = 44) and 3′ (lower: WT, n = 167, ∆5′/∆3′ kb: n = 34) ends to the core and shell or the shell in WT and ∆5′/∆3′ kb cells treated with MG132 (5 μM for 6 h). D (upper) EM observation of the paraspeckles in MG132-treated (5 μM for 17 h) ∆5′/∆3′ cells using NEAT1_5′ (left) and NEAT1_D2 probes (right). Scale bar, 100 nm. (middle) Graph showing the proportion of the localization of NEAT1_5′ (left, 310 gold particles) and NEAT1_D2 (right, 294 gold particles) probes within the paraspeckles in HAP1 NEAT1 ∆5′/∆3′ cells. (lower) Graph showing the proportion of the localization of NEAT1_5′ (left, 19 cells) and D2 (right, 22 cells) probes in each paraspeckle in ∆5′/∆3′ cells. Each box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top). E Schematics of the NEAT1_2 configuration in the WT and ∆5′/∆3′ mutant.
Figure 1(A) Yeast two-hybrid assays to test for interaction between Munc18-1 and ERK1/2. pGAL4-Munc18-1 (M18) and pGAL4-Empty (control) were used as baits and ERK1 and ERK2 as prey. Both ERK1 and ERK2 bind Munc18-1 and allow growth on selection plates (minus Histidine (-HIS) and minus Histidine and Adenosine (-ADE)). (B) In vitro kinase assays showing Munc18-1 phosphorylation by ERK. Radioactive phosphoblot (32P) and immunoblot (IB) of in vitro ERK kinase assay using immunoprecipitatedMunc18WT, Munc184A (M18T78A,S158A,S241A,T574A), Munc183A (M18S158A,S241A,T574A), and Munc18AA (M18S241A,T574A) from HEK293T cells incubated with recombinant ERK2. The phosphoblot shows phosphorylated Munc18-1 (pMunc18-1) and auto-phosphorylated ERK2 (pERK2).(C) Quantification of phospho-Munc18-1 intensity in wild-type and Munc18AA mutant expressed as the amount of 32P incorporation in Munc18-1 normalized to total Munc18-1 levels. Phosphorylation of Munc18WT was set to 100%. (Munc18WT: 100±3%; Munc18AA: 24±2%, n=3, * p = 0.01). (D) ERK1 in vitro kinase assay showing ERK-dependent 32P-incorporation in Munc18WT immuno-precipitated from HEK293T cells. Western Blot stained with Munc18-1 antibody shows total precipitated Munc18-1 levels were identical in all conditions. (E) Alignment of amino acid sequence of Munc18-1 in different species. ERK consensus site (P-X-S/T-P) (yellow) around phosphorylation site S241 (red) is highly conserved. (F) Three-dimensional structure of Munc18-1 (blue, green and yellow ribbons) and syntaxin 1 (purple and red ribbons). Zoom (right) of grey box in full structure (left) illustrates position of the ERK phosphorylation sites S241 (red) and T574 (blue). The T574 site has been reported as phosphorylation site for Cdk5 (Shuang et al, 1998).
H3K122succ peaks localize to gene promoters.Figure 1A Peptide dotblot array probed with anti-H3K122succ AB #2. Peptides with indicated modifications were spotted on membranes. B Immunoblot analysis with anti-H3K122succ AB #2 on indicated histone octamers. Octamers with H3K122succ are specifically recognized. C Pairwise scatterplots of H3K122succ read counts within peaks from ChIP experiments using all three H3K122succ antibodies (AB #1, AB #2 and AB #3). D Genomic feature distribution of H3K122succ peaks obtained with the three H3K122succ antibodies. The percentage of peaks in the 1st exon, remaining exons, intergenic regions, 1st intron, other introns, promoter/ TSS (≤ 1 kb from TSS), promoter (1-2 kb) and 3' UTR is displayed. A random distribution of peaks is shown for comparison
Figure 4(A) Scheme of affinity purification technique to isolate components that bind Akt. Four immunoprecipitation experiments of USP1, PHLPP1, UAF1 and FoxO from muscle homogenates during fasting were performed in parallel to non-specific mouse IgG control, and protein precipitates were analyzed by mass spectrometry. (B) Mass spectrometry data obtained in (A) is presented using Venny diagram tool. Only proteins that were at least threefold more abundant in the immunoprecipitation samples than in IgG control are listed. (C( In fasting, Akt sediments to glycerol gradient fractions containing USP1/UAF1, PHLPP1, and TSC1/TSC2. Muscle homogenates from fed or fasted mice were separated on 10%-40% glycerol gradient, and alternate fractions were analyzed by SDS-PAGE and immunoblot. Red rectangle marks fractions containing USP1/UAF1, PHLPP1, TSC1 and TSC2 in fed conditions. (D) During fasting, Akt is recruited to USP1-PHLPP1-TSC1 complex. TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or USP1(C90S) from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Black lines indicate the removal of intervening lanes for presentation purposes. (E) During fasting, inhibition of USP1 reduces rates of protein synthesis. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM. (F) Inhibition of USP1 results in TSC1 accumulation. Soluble fraction of muscles expressing shLacz or USP1(C90S) from fasted mice were analyzed by SDS-PAGE and immunoblotting. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz. Data is presented as protein content (AU) in USP1(C90S) vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM. (G) mTOR inhibition with rapamycin results in reduced USP1 protein content and enhanced Akt phosphorylation at T308. However, a simultaneous downregulation of USP1 is required to promote TSC1 accumulation. Soluble fraction of muscles expressing shLacz or gUSP1 (in vivo CRISPR) from fed and fasted mice injected i.p. with rapamycin (6mg/kg body weight) or saline were analyzed by SDS-PAGE and immunoblot.
Figure 3(A) Representative confocal images of a hippocampal neuron (DIV14) immunostained for MAP2, β-III-tubulin and DCLK1. Scale bar = 20 µm (B) Quantification of the average ratio of DCLK1, MAP1A, MAP1B and MAP2 to total tubulin immunofluorescence intensities in dendrites compared to axons. Per condition 6-9 cells were analyzed. Error bars indicate SEM. (C) Representative images of DIV10 hippocampal neurons transfected for 4 days with plasmids encoding β-gal for visualizing the transfected cells and three different DCLK1 shRNAs or DCLK2 shRNA and immunostained for β-gal (red) and DCLK1 (green). (D) Quantitative analysis of normalized average intensity of DCLK1 in regions of interest of DIV10 neurons transfected with DCLK1 sh#1 (n = 16 neurons), DCLK1 sh#2 (n=16) and DCLK1 sh#3 (n=18). Non-transfected cells were taken as a control (n = 48). N=2. Error bars indicate SEM; *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test). (E) Example of Western blot analysis of protein extracts of DIV4 cortical neurons electroporated on DIV0 with pSUPER (control) or pSUPER encoding one of three DCLK1 shRNAs. Levels of actin were used as an equal loading control. (F) Quantification of protein levels of DCLK1 of samples represented in (E). N = 3, error bars indicate SEM, *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test). (G) Representative maximum projections of peroxisome distribution in DIV14 neurons transfected with PEX-mRFP-FKBP, KIF1C (MDC)-FRB and the indicated shRNA before and 30 minutes after rapalog addition. BFP fill was co-transected to visualize the morphology of transfected neuron. Axons are marked with a blue line. Arrows mark dendrite (gray) and axon (blue) targeting of peroxisomes after rapalog addition. Scale bar = 20 µm (H) Quantitative representation of the percentage of DIV14 neurons transfected with PEX-RFP together with either KIF1C, KIF21A or KIF5B and an indicated shRNA. as in (A) in which after addition of rapalog peroxisomes redistributed to axons ("a", blue bars), to both axons and dendrites ("a+d", red bars) or did not redistribute into any neuronal compartment ("nt"-no targeting, gray bars). Per condition 19-44 neurons were analyzed; N = 2.
CIP2A expression correlates with mTORC1 activity in primary human breast tumors.Figure 4.CIP2A expression correlates with mTORC1 activity in primary human breast tumors. TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores (arbitrary units [a.u.]) for duplicate samples were averaged and plotted according to mean CIP2A expression (left). The red lines are the regression lines, and the stippled red lines define the 95% confidence intervals. Statistical analysis of data was performed using NCSS 2007 and Prism version 5. Examples of the staining of breast cancer (a) and normal (b) cores indicated in the P-T389-S6K1/CIP2A plot are shown on the right. rsp, Spearman's correlation coefficient.
Spitz from photoreceptors is transported to the lamina and is required for lamina glia maintenance.Fig 5(A, A') Anti-Spitz (red) immunostaining of w1118 adult head. Spitz can be detected in the retina and lamina. (B, B') The full-length transmembrane form of Spitz-GFP (mSpi, green) expressed in the retina in GMR>mSpiGFP flies was predominantly identified in the photoreceptor soma and terminally localized in the lamina cartridge. mSpitz-GFP requires processing by Rho and Star to become a secreted form. Overexpressed mSpitzGFP has been demonstrated to be retained in the perinuclear ER even in the presence of endogenous Rho/Star [56] (C) Knockdown of both EGFR ligands Spi and Krn in R1-6 photoreceptors in Rh1>Dcr2+Spi-RNAi+Krn-RNAi and (D) blockade of Spi processing in Rh1>lacZ+iRhom exhibited lamina degeneration after shifting to 29°C for 14 days. (E-E') Lamina degeneration in whole eye rho7M43ru1 double mutant clones at 28°C for 12 days. The clone is labeled by DsRed (red). (F) Spi secretion is inhibited in Rh1>lacZ+Rab11S25N. (G) The percentages of the vacuole areas of (C, D, F) in lamina at 29°C for 14 days were examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. DAPI: nuclei (white in A', B', C-F). Scale bar: 20 μm.
Figure 1(A, B) Knockdown of hnRNPA3 increases poly-GA expression while expressions of EGFP protein levels are not altered upon knockdown of hnRNPs. The control ("x0") vector lacks the G4C2 repeats but still contains the 5' flanking region and 3x TAG. ANOVA, Dunnett. (C, D) Overexpression of hnRNPA3 suppresses poly-GA expression. n=2 experiments performed in duplicates. ANOVA, Tukey. (E) Increased repeat RNA upon hnRNPA3 knockdown. EGFP RNA levels are only slightly increased as compared to levels of the repeat RNA. n=2 experiments performed in duplicates. ANOVA, Dunnett. (F, G) Knockdown of hnRNPA3 increases RNA foci. n=3 replicates; two tailed paired t-test, Scale bar=20 μm. All graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.