EMBO/sd-character-level-ner huggingface dataset:
Figure 4Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (A) CIV subunits COX1 and COX5B. (B) CI subunit NDUFB7. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control. Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (B) CI subunit NDUFB7. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control. Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (C) CIII subunit CORE2. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control. (D) Structural arrangement of COX subunits detected in association with SC I+III2. Upper illustrations depict lateral views of human SC I+III2+IV1, where CI (aquamarine) and CIII2 (light green) are positioned along the inner membrane plane (dots). CIV appears as a smooth backbone with the associated COX subunits highlighted to show their positions relative to SC I+III2: COX7A2 (blue) and COX5B (red) attach COX1 (yellow) to the concave surface formed by CIII2 and the CI membrane arm, interaction further stabilized by COX7C (green) binding to CI. COX4 (magenta) interacts with COX1 on the distal side of CIV. Medium illustrations: matrix (M) view. Lower illustrations: intermembrane space (IMS) view. The figure was created with USCF Chimera v.1.12 using the 5XTH structure (Guo et al., 2017) from the PDB database.
Figure 7A: Presence of putative AREs (AUUUA) in transcripts of WT oocyte. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (MII/GV) in WT oocytes B: Presence of putative AREs in oocyte transcripts stabilized by Cnot6l-/- knockout. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (Cnot6l-/-/WT) at MII stage C: Co-IP results showing interaction of ZFP36L2 with CNOT6L and CNOT7. HeLa cells were co-transfected with plasmids expressing HA-ZFP36L2 and FLAG-CNOT6L/7 for 48 h before immunoprecipitation. Experiments were performed three times with reproducible results; a representative result is shown D: RNA immunoprecipitation results showing interaction of CNOT6L with indicated transcripts, with or without the presence of ZFP36L2. HeLa cells were co-transfected with plasmids expressing FLAG-ZFP36L2 and HA-CNOT6L for 48 h before immunoprecipitation using an anti-HA antibody. mRNAs recovered from the immunoprecipitates were subjected to qRT-PCR. Error bars, standard deviations (n = 3 biological repeats). ***P < 0.001 by two-tailed Student"s t
Figure 2U2OS cells were lysed and the lysates were incubated with anti-PRMT5 antibody or immunoglobulin G (IgG) as control followed by protein A Sepharose bead addition. The immunoprecipitated (IP) proteins or whole cell extract (WCE) were separated by SDS-PAGE and Western blotted (WB) with the indicated antibodies. Same as panel (A) except anti-DDX5 antibody was used instead of anti-PRMT5 antibody for immunoprecipitation. The mass spectrum of R502 monomethylation from Flag-DDX5 expressed in U2OS. HEK293 cells transfected with Flag-DDX5 were lysed and immunoprecipitated with anti-Flag antibodies. The bound proteins were along with WCE from untransfected (-) and Flag-DDX5 (+) transfected cells were Western blotted with anti-FLAG, anti-methylarginine antibodies (MeR). The knockdown using siRNAs of the indicated gene in U2OS cells was confirmed by Western blotting The knockdown using siRNAs of the indicated gene in U2OS cells were analyzed by immunofluorescence with S9.6 and anti-nucleolin antibodies. A typical image is shown.
ActivinB signaling is a potential therapeutic target for patients of group 3 MB.Figure 7(A) Kaplan-Meier representing survival of mice treated with either vehicle (black) or Galunisertib (LY2157299, red) or Cisplatin (blue) or a combination of Galunisertib and cisplatin (purple) after orthotopic grafting of PDX4 cells into the cerebellum. The pink rectangle represents Galunisertib treatment duration, while the blue dotted lines represent the 3 cisplatin administrations. The p-values were determined by Log-Rank (Mantel-Cox) test on panel A *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3. (B) Boxplot of tumor area after 25 days of treatment. On the right, boxplots represent quantification of P-Smad2 staining on tumor (IHC). The p-values were determined by unpaired t-test on panel B. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3. (C) P-Smad2 staining by IHC in 3 representative tumors per group after 25 days of treatment. The scale represents 500μm and 100μm on the left and right panels, respectively. (D) Boxplots representing the expression level of INHBB, PMEPA1, MYC and NRL in the different MB subtypes, as defined in Cavalli et al. (Cavalli et al., 2017). Only p-values corresponding to comparisons between Group 3 subtypes are indicated. Patient samples are colored by subtypes as indicated. (E) Scatter plot of INHBB and PMEPA1, expression levels in Group 3 patient samples. Colored dots represent each patient sample and colors represent the group 3 MB subtypes (α in yellow, β in brown and δ in orange). Wilcoxon rank-sum tests were performed for panel D. Spearman's rank correlation coefficient ρ and p-value are indicated on panel E. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.
Figure 1Western blot analysis of Ronin depletion upon treatment of Ronin fl/del Cre-ERT2 cells with 4OHT. Brightfield images of Ronin fl/del and Ronin del/del ESC. Cell count of Ronin fl/del and Ronin del/del ESC. Data represent mean ± SD, n = three independent experiments. Annexin V cell death assay of Ronin fl/del and Ronin del/del cultured for 6 days. Two-tailed unpaired Student's t test, data represent mean ± SD, n = three independent experiments. FACS plot of cell cycle distribution of day 6 Ronin del/del and Ronin fl/del, determined by EdU assay. Quantification of the cell cycle distribution determined by EdU assay. Data represent mean ± SD, n = three independent experiments. Ronin fl/del and Ronin del/del ESC stained for Ronin (left panel) or Oct4 (right panel). The nuclei are counterstained with DAPI.
Figure 5Apo-Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations (2.5 mM) of cAMP, cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP (5 mM) was measured spectrophotometrically. Values are the average of three repeats, error bars represent standard deviation. HPLC traces of reaction products generated from 100 µM c-di-GMP incubated with apo-Bd1971 (Control), or protein supplemented with either 100 µM cAMP or cGMP. Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure.
Figure 5U2OS cells were transfected with a Flag-DDX5 expression vector in the presence of siCTL or siPRMT5. Lysates were immunoprecipitated with IgG or anti-Flag antibodies as indicated. The WCE and the bound proteins were Western blotted with anti-symmetrical dimethylarginine antibody or anti-Flag antibodies. A schematic of DDX5 helicase domain and RGG/RG motifs is shown. GST-fusion of DDX5 fragments (F1-F4) and the RK mutant used in this study. RK (*) indicates R to K substitutions of 5 arginines (R478, R482, R484, R486, R502). Coomassie blue (left panel) and in vitro methylation assay (right panel) of indicated GST-DDX5 fragments and the GST-DDX5-RK mutant. Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with Flag-tagged DDX5, DDX5-RK or DDX5-XD (helicase dead). Nuclear S9.6 signal was counted in both Flag-negative and Flag-positive cells. The Flag-negative cells were considered as untransfected cells. The graphs shown represent the quantification with the S.E.M from 3 independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001. Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with siCTL or siDDX5-1 and Flag-tagged DDX5 (+WT) or DDX5-RK (+RK) as indicated. The graphs show the average and S.E.M. from three independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001. HEK293 cells were transfected with siCTL, or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). Forty eight hours later, all the cells were subjected to DRIP-qPCR analysis. The average with the S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and n.s.: not significant.
Gene expression analyses of E19 organoids at early and late culture time points.Figure 1Fetal organoids isolated from fetal intestine at embryonic day 19 were cultured for 30 days in ENR medium and analysed 3 days after indicated passage. PCA was conducted on global gene activity in mouse fetal tissue at day 0 and 42, mouse E19 organoids at day 3 and 30 of culture, mouse adult organoids at day 3 and 30 of culture (n=4 independent intestinal tissue specimens and n=4 independent organoid cultures). Lists of differentially expressed genes between adult and fetal tissue, day 3 and 30 organoids were generated for C up and D down regulated genes. Results are shown as Venn diagram. Curated heatmaps of top 76 up and 72 down regulated genes. Highlighted genes were chosen based on biological interest. The coloured bar represents the expression level from low (green) to high (red). The most significantly changed canonical pathways between fetal organoid at day 3 and 30 of culture.
Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.f3Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet (LFD)-kept mice (n=5) of same age were used as a negative control. (a) Body weight was daily monitored during injection period. (b) Daily food consumption was measured during injection period. (c) Livers were collected from indicated mice and photographed. (d,e) Total liver mass (d) were measured from indicated mice. (c) Livers were collected from indicated mice and photographed. (d,e) Total liver mass (d) and total epididymal white adipose tissue (eWAT) mass (e) were measured from indicated mice. (f-h) Relative mRNA expression was analysed from eWAT of indicated mice through quantitative reverse transcription-PCR. (i,j) Liver sections were analysed by hematoxylin and eosin (HE, upper panels) and Oil Red O (ORO, lower panels) staining (i). ORO densities were quantified (j). (k,l) Calcium-induced CaMKII autophosphorylation in livers was analysed by immunoblotting (k) and quantified (l). (m) Primary hepatocytes from 2-month-old C57BL/6 mice kept on LFD were treated with bovine serum albumin (Con), PA (500 μM), PA+verapamil (Ver, 50 μM) or PA+nicardipine (Nic, 100 μM) for 12 h. After each treatment, cells were loaded with a calcium indicator Fura-2-AM. Calcium levels were visualized by dual fluorescent microscopy at 340/380 nm, and ratio of fluorescence intensities of Fura-2-AM at 340 nm over 380 nm was quantified (n=17). Scale bar, 1 cm (c), 200 μm (i). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.
Figure 9Systolic, diastolic and mean blood pressure in Coq9+/+ and Coq9R239X mice (A). Heart rate in Coq9+/+ and Coq9R239X mice (B). **P < 0.01; Coq9R239X mice versus Coq9+/+ mice (t test; n = 5 for each group).
Figure 5Immunoblot analysis of whole cell lysate from untreated human THP-1 monocytes with stable expression of a non-targeting control shRNA or and shRNA targeting OTULIN. Data are representative of two independent experiments. Immunoblot (left) and densitometry (right) analysis of IκBα levels in shControl and shOTULIN THP-1 cells treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments. ELISA analysis of spontaneous TNF secretion over 96 h in shControl and shOTULIN THP-1 cells. Data represents mean ± SEM (n=4) and were analysed using the two-way ANOVA test of statistical significance with Sidak's correction for multiple comparisons. Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in shControl and shOTULIN THP-1 cells in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments. Densitometry analysis of p65/RelA phosphorylation as presented in (D) from three independent experiments.
Figure 6A Confocal immunofluorescence microscopy images of freshly purified pDCs co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 5 µm. B Confocal immunofluorescence microscopy images of pDCs incubated for 24 h with TGF-β or IFN-γ and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). Scale bar, 5 µm. C Pearson's coefficient of IDO1 colocalization with EEA1 in pDCs either untreated or stimulated with TGF-β or IFN-γ (as in B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from three independent experiments).
Fig.Figure 3A) Schematic representation of loading and purification of PCNA-Ub on nicked circular DNA. The DNA-loaded PCNA-Ub elutes in the void of the SEC column and this sample is collected and used for studying activity of USP1 on DNA-loaded PCNA-Ub. B) Michaelis-Menten analysis of USP1+UAF1 with and without DNA (65bp dsDNA) shows that DNA binding alone has no effect on USP1 activity (n=2, technical replicates (example shown here from 1 of 2 biological replicates)). C) Coomassie stained gel of in vitro activity assay showing increased activity of USP1-UAF1 on DNA-loaded PCNA-Ub compared to PCNA-Ub (-DNA) and PCNA-Ub (+DNA, +RFC and not ATP). D) Quantification of gel-based activity assays showing enhanced activity of USP1+UAF1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)). E) Quantification of gel-based activity assays showing enhanced activity of USP1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).
Fig.Figure 3a, CPAP co-immunopurifies with CDC components from HeLa extracts. Centrosomal proteins Cap350 and ODF2 do not co-purify with CPAP. b, Levels of CDC components (CPAP, Aurora A, OFD1, and Nde-1) are differentially regulated in response to serum starvation and stimulation in WT and Seckel fibroblasts. Specifically, they are elevated 1h after serum stimulation, thus enabling cilium disassembly. Elevated CDC levels in WT cells correlate with an elevated level of CPAP as shown in Fig. 2 as well as with the expression of cyclin A, a G1-S marker. GAPDH was used as a loading control indicating an equivalent amount of WT and Seckel fibroblast total proteins were loaded. c. WT CPAP (WT-cells IP) but not Seckel variant CPAP (Seckel cells IP) co-immunopurifies with CDC components from asynchronously grown WT and Seckelfibroblastscell extracts. The IP samples were adjusted so that equivalent amounts of immunoprecipitatedCPAP were used in samples. Seckel (5x) and WT (1x) fibroblast cell extracts were used as input controls to show the efficacy of the various antibodies. Note that CPAP abundance in Seckel cells is ~5 times lower than that of WT cells. CentrosomalproteinsCap350 and ODF2 do not co-purify with CPAP indicating the specificity of CPAP interaction with CDC components. d, GST pull down (i) and immunopurification assays (ii) of CPAP's CC5 domain demonstrate its sufficiency in mediating CPAP-CDC protein interactions. The CC5 fragment is expressed as a GFP fusion protein in HeLa cells and immunopurified using anti-GFP antibodies. HeLa extracts were used as input controls. CAP350 does not co-purify with CPAP. Both Cap350 and ODF2 are not pulled down by CPAP's CC5 domain.
Comparative phosphoproteomics to identify processes regulated by PfCDPK7A.Figure 1A. Comparative phosphoproteomic and proteomic analyses were performed on late ring / early trophozoite stage wild type (3D7) or PfCDPK7-KO parasites. The phosphorylation fold-change ratio of identified phosphopeptides was normalized to total protein abundance fold-change. The fold-change ratios for all phosphopeptides from various replicates are provided The S-curve for the normalized data is provided for some of the significantly altered hyper-(blue) and hypo-(red) phosphorylated sites belonging to key proteins are indicated. Phosphorylation sites of key enzymes involved in PL biosynthesis that exhibited reduced phosphorylation in PfCDPK7-KO parasites are indicated in green. B. Pathway analysis of proteins that exhibited reduced phosphorylation upon PfCDPK7 disruption and the possible metabolic pathways these proteins may regulate The pathways are represented based on their p-values (log 2), as estimated by the gene ontology tool available at PlasmoDB. C. Protein-protein interactions were predicted between differentially phosphorylated proteins using the STRING resource. The analysis exhibited high confidence interactions between the candidate proteins A major signaling module that involved enzymes including EK and PMT implicated in PC/PE metabolism was identified, in addition to a network involving trafficking protein PfRab11b. Hyper- and hypo-phosphosphorylated proteins are indicated in blue and red respectively. Proteins that are relevant to the topic of this paper are encircled. Proteins involved in PL metabolism and trafficking are indicated in red and blue, respectively.
Figure 1(A) flg22-induced MAPK activation in Col-0 and fer-4 mutant roots under Pi-starvation conditions. Seedlings grown in either HP or LP media were treated with 1 μM flg22 for 15 mins and roots were harvested. The MAPK level was determined with anti-pMAPK, and β-actin was used as a loading control. Data information: All experiments were repeated three times with similar results.  (B) flg22-induced ROS production in Col-0 and fer-4 mutant roots under Pi-starvation conditions. Seedlings grown in either HP or LP media were treated with 1 μM flg22, RALF23 or both flg22 and RALF23 for 15 mins. Roots were harvested and stained with H2DCFDA. One representative root was shown. (C) Average H2DCFDA signal intensity in roots shown in (B). The fluorescence intensity was quantified with ImageJ. The data shown indicate the means ± SDs (n = 30, n refers to the number of roots per group); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.  (D) Heatmap of differentially expressed defense-responsive genes after LP or RALF23 treatment (n = 3, n refers to technical replicates). Seven-day-old Col-0 seedlings were treated with either LP for 5 days or 1 μM RALF23 for 2 hours. Data information: All experiments were repeated three times with similar results.  (E) Density figure showing the comparison of defense-responsive genes between LP treatment and RALF23 treatment (n = 3, n refers to technical replicates). The x- and y-axes represent changes in transcription level, and the red area indicates high density. Spearman correlations were used for the correlation analysis. Data information: All experiments were repeated three times with similar results.  (F) RALF23 inhibits flg22-induced MAMP-responsive FRK1 gene expression in a Pi content-dependent manner. Seedlings grown in either HP or LP media were treated with water or flg22 for 15 mins and roots were harvested. The relative expression of FRK1 was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.  (G) Growth of Pto DC3000 in Col-0 and RALF23-OE lines under Pi-starvation conditions. Seedlings grown in either HP or LP media were root soaking inoculated with bacterial suspension (OD600 = 0.2) for 2 min, and the number of bacteria from individual roots was quantified at 0 dpi and 3 dpi. The data shown indicate the means ± SDs (n = 27, n refers to the number of roots per group); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.