Journal: Nature Communications

Article Title: The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors

doi: 10.1038/s41467-022-33433-3

Figure Lengend Snippet: a An illustration of the Pol II-mediated clusters. RUVBL1/2 were identified by Pol II, H3K27ac, and H3K4me3 ChIP-MS. b RNA polymerase I/II/III subunits were detected in RUVBL2 ChIP-MS preparations. The lines indicate the protein-protein interactions detected in the STRING database. c Heatmap illustrating that RUVBL1 and RUVBL2 globally colocalized with Pol II on the mESC genome. d Schematic diagram showing the subcellular fractionation (cytoplasm, nucleoplasm, and chromatin) procedure, which was adopted from Damianov et al. . e Western blotting was performed to examine the different subcellular fractions of mESCs. a-Tubulin, histone H3, and snRNP70 were used as marker proteins of the cytoplasm, chromatin, and nucleoplasm, respectively. WCE indicates whole-cell extracts. f Chromatin fractions in wild-type mESCs were separated through size exclusion chromatography, and western blotting was performed to identify RPB1, RPB7, RUVBL1, and RUVBL2 in the gradient eluents based on their different molecular weights. g Protein-protein interactions between Pol II (RPB1) and RUVBL1 or RUVBL2 were examined under different concentrations of NaCl. h The phosphorylation states of Pol II interacting with RUVBL1/2 using RUVBL1 and RUVBL2 degron mESCs were examined. RPB1-CTD or RPB1-Unp indicates the unphosphorylated RPB1 CTD, and RPB1-S2P and RPB1-S5P indicate RBP1 phosphorylated at serine 2 and serine 5 in the CTD, respectively (lower panel). i The RPB1 CTD was acutely degraded, and the native chromatin fractions were subjected to an antibody recognizing the NTD of RPB1 during immunoprecipitation. j Schematic diagram showing the method used to confirm that RUVBL1/2 interacted with RPB1 CTD on chromatin (upper panel). The ChIP-qPCR data shown represent 3 biological replications. Two-tailed Student’s t tests were performed to determine the significance of differences between groups, data are presented as mean +/- SDs (ns, not significant, * p < 0.05, *** p < 0.001, **** p < 0.0001), the exact p values can be found in the source data.

Article Snippet: The IPed samples were analyzed with RPB1-CTD (Abcam, ab26721, dilution 1:3000) antibodies (to confirm the Pol II degradation), RPB1-NTD antibodies (dilution 1:3000) and RUVBL1 (Santa cruz, sc393905; dilution 1:2000) or RUVBL2 antibodies (Santa cruz, sc374135, dilution 1:2000), and RPAP3 (ABclonal, A15239; dilution 1:2000).

Techniques: Protein-Protein interactions, Fractionation, Western Blot, Marker, Size-exclusion Chromatography, Phospho-proteomics, Immunoprecipitation, ChIP-qPCR, Two Tailed Test

Journal: Nature Communications

Article Title: The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors

doi: 10.1038/s41467-022-33433-3

Figure Lengend Snippet: a Schematic showing the system used for simultaneous imaging of tethered proteins and recruited proteins, as published previously . b Representative images of endogenous RUVBL1 and RUVBL2. Scale bar, 5 μm. c The relative IF signals of the target proteins at the LacO loci under mock (RUVBL1 n = 24, RUVBL2 n = 30) and Dox treatments (RUVBL1 n = 18, RUVBL2 n = 30). Box plots indicate median (middle line), 25th and 75th percentile (box) and 5th and 95th percentile (whiskers), as well as outliers (single black points), differences between groups were analyzed by two-tailed Student’s t tests (**** p < 0.0001). d The mean and total intensity of LacR-BFP fusion signals at LacO loci per cell were plotted in bar graphs. The cotransfected cells ( n = 30) in different fields from 3 transfection replicates were used for quantification. Differences between groups were analyzed by two-tailed Student’s t tests. “ns” indicates no significant difference. The error bars indicate mean + /− SDs. e Representative images of endogenous Pol II before and after transcriptional activation. Scale bars, 5 μm. f The relative IF signals of Pol II and LacR-BFP fusions at the LacO loci were plotted as boxplot. Cells from a minimum of 10 different fields (BFP n = 11, RUVBL1 n = 28, RUVBL2 n = 29 in mock state, and BFP n = 13, RUVBL1 n = 24, RUVBL2 n = 16 in Dox activation state) were used for quantification. Differences between groups were analyzed with two-tailed Student’s t tests (ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). g Nuclear Pol II (RPB1) density was quantified from 3 biological replicates. Two-tailed Student’s t tests were performed to determine the significance of differences between groups, and the error bars indicate the mean + /− SD. h IF assay was performed to examine endogenous RUVBL2. Cells (BFP n = 22, CTD n = 19) from a minimum of 10 different fields were used for clock scanning and quantification, and the error bars indicate the mean + /− SDs. The clock scan densities were analyzed with two-tailed Student’s t -tests (**** p < 0.0001). Scale bars, 5 μm.

Article Snippet: The IPed samples were analyzed with RPB1-CTD (Abcam, ab26721, dilution 1:3000) antibodies (to confirm the Pol II degradation), RPB1-NTD antibodies (dilution 1:3000) and RUVBL1 (Santa cruz, sc393905; dilution 1:2000) or RUVBL2 antibodies (Santa cruz, sc374135, dilution 1:2000), and RPAP3 (ABclonal, A15239; dilution 1:2000).

Techniques: Imaging, Two Tailed Test, Transfection, Activation Assay

Journal: Nature Communications

Article Title: The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors

doi: 10.1038/s41467-022-33433-3

Figure Lengend Snippet: a Schematic diagram showing the RPB1 CTD and RUVBL1/2 pull-down assays. b The complexes pulled down by beads were analyzed by western blotting. c RUVBL1/2 promoted RPB1 CTD clustering in vitro. The scale bar is 20 μm. d Plot showing the cluster area described in ( c ), clusters larger than 100 square pixels were counted (Mock n = 139, RUVBL2 in order n = 300, 71 and 39, respectively) from no less than 8 different fields for each condition. Two-tailed Student’s t tests were performed to determine the significance of differences between groups, and the error bars indicate the mean + /− SD (**** p < 0.0001). e Plot showing the condensed fraction in the different fields per condition presented in ( d ). Two-tailed Student’s t tests were used to determine the significance and the data are reported as the mean + /− SD (* p < 0.05, *** p < 0.001). f Schematic diagram showing the in vitro Pol II transcription initiation system. g Nascent RNA synthesis by the preinitiation complex on the HDM2 promoter. This result represents 3 replicates. h In vitro droplet assay with GFP-EWS-FLI1 and RPB1 CTD-mCherry. i Boxplot showing the droplet area of mCherry signals. The total number of droplets examined in one in vitro droplet experiment was n = 127 under the mock condition and n = 197 under the RUVBL1/2 addition condition. The bottom edge of the box represents the 25th percentile, and the top represents the 75th percentile. Most data points are covered by whiskers (1.5x interquartile range). Two-tailed Student’s t tests were performed to determine the significance of differences between groups (**** p < 0.0001). j Strategy for detecting EWS-FLI1 condensates at sites of DNA and RPB1 CTD recruitment by RUVBL1/2 . k Wide-field total internal reflection microscopy (TIRFM) images of EWS-FLI1 and RPB1 CTD N26 -mCherry in DNA Curtain experiments. l The efficiency of RPB1 CTD N26 -mCherry recruitment as described in ( j ). Three independent DNA curtain experiments were repeated. The error bars refer to mean + /− SD. Statistical significance was evaluated by two-tailed Student’s t test (** p < 0.01); confidence level: 95%.

Article Snippet: The IPed samples were analyzed with RPB1-CTD (Abcam, ab26721, dilution 1:3000) antibodies (to confirm the Pol II degradation), RPB1-NTD antibodies (dilution 1:3000) and RUVBL1 (Santa cruz, sc393905; dilution 1:2000) or RUVBL2 antibodies (Santa cruz, sc374135, dilution 1:2000), and RPAP3 (ABclonal, A15239; dilution 1:2000).

Techniques: Western Blot, In Vitro, Two Tailed Test, Microscopy

Journal: Nature Communications

Article Title: The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors

doi: 10.1038/s41467-022-33433-3

Figure Lengend Snippet: a Cell growth analyses after RUVBL2 depletion ( n = 3 biological replicates), error bar indicates the mean + /− SD. b Diagram showing the experimental design for the treatment of RUVBL2-degron cells. c Western blot analyses of RUVBL2 protein levels at different time points during RUVBL2 degradation. d Snapshots of poly(A) RNA-Seq signals on the c-Myc, Ccnd1, Chop, and Atf4 loci at different time points during RUVBL2 degradation. e Cluster analyses of RNA-Seq expression signals after RUVBL2 degradation at different time points. The enriched GO terms and the motifs were shown accordingly. f The results of the metagene analyses of RUVBL1 and RUVBL2 ChIP-Seq signals at the gene promoters of each cluster are shown in e . g Pie charts showing the distribution of target genes corresponding to specific transcription factors peak belonging to the clusters in e . The “other” cluster indicates the targeted genes that did not belong to any other cluster. “Random” indicates the distribution of 8000 genes randomly selected from the genome. h The numbers of genes in each cluster shown in e that were simultaneously affected by UPF1 knockdown are plotted in a bar graph. The percentages in the bracket were the ratio of the number of genes in each cluster as shown in e . i Cluster analyses of the percentage splicing index (PSI) after RUVBL2 degradation at different time points. The corresponding enriched GO terms are shown. j The PSI of Smg1 exon 2 is shown on bar graphs. Error bars indicate the mean + /− SDs. rMATS were used to calculate the significance difference of PSI ( n = 2, * p < 0.05, ** p < 0.01, *** p < 0.001). The position of the exon and the splicing event are illustrated in the upper panel. The FDR are extracted from rMATS by adjusting p-values with BH method.

Article Snippet: The IPed samples were analyzed with RPB1-CTD (Abcam, ab26721, dilution 1:3000) antibodies (to confirm the Pol II degradation), RPB1-NTD antibodies (dilution 1:3000) and RUVBL1 (Santa cruz, sc393905; dilution 1:2000) or RUVBL2 antibodies (Santa cruz, sc374135, dilution 1:2000), and RPAP3 (ABclonal, A15239; dilution 1:2000).

Techniques: Western Blot, RNA Sequencing, Expressing, ChIP-sequencing, Knockdown

Journal: Nature Communications

Article Title: The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors

doi: 10.1038/s41467-022-33433-3

Figure Lengend Snippet: a Workflow of the process used to identify the genes directly targeted by RUVBL2. The FDR are extracted from DESeq2 by adjusting p-values using BH method. b Heatmap analyses of RUVBL2 direct target genes based on PRO-Seq expression. c Snapshots showing the RUVBL1 and RUVBL2 ChIP-Seq signals and nascent RNA (PRO-Seq) signals at the Bmp4 and c-Myc loci after RUVBL2 degradation. d ChIP-Seq signals in the promoter regions of 45 genes directly targeted by RUVBL2 and 45 nontargeted genes in 153 datasets obtained from the Cistrome DB (Supplementary Data ). e The 10 factors with the largest and smallest coefficients in the elastic net were selected. The heatmap shows the relative occupancy of these 10 factors in the 45 genes directly targeted by RUVBL2 and in 45 nontarget genes. f Pie chart indicating the percentage of C-MYC or CXXC1 ChIP-Seq-determined target genes that overlap the RUVBL2-affected genes as determined by the RNA-Seq data shown in Fig. . g Profiles of PRO-Seq signals at the gene regions of directly targeted genes ( n = 45), all genes ( n = 36821) and posttranscriptional early response genes ( n = 41) after RUVBL2 degradation. The profile above 0 indicates the signal on the sense strand, the profile below 0 indicates the signal on the antisense strand. Two-tailed Wilcoxon tests was performed to calculate the significance (* p < 0.05, ** p < 0.01, *** p < 0.001). The center line of boxplot represents median, the box limits represent upper and lower quartiles, and the whiskers represents 1.5x interquartile range or maximum/minimum value. h The RUVBL2 direct target gene Bmp4 was examined by single-molecule RNA FISH combined with RPB1 immunofluorescence. Representative images are shown, scale bar is 3 μm. i The signals of the Pol ll (RPB1 IF) cluster that overlap with nascent RNA foci were measured, the boxplot indicated median (middle line), 25th and 75th percentile (box) and 5th and 95th percentile (whiskers), as well as outliers (single black points). Statistically analyzed (two-tailed Student’s t test) from at least 10 different fields of each condition (Auxin 0 h n = 61, Auxin 1 h n = 38). ** p < 0.01.

Article Snippet: The IPed samples were analyzed with RPB1-CTD (Abcam, ab26721, dilution 1:3000) antibodies (to confirm the Pol II degradation), RPB1-NTD antibodies (dilution 1:3000) and RUVBL1 (Santa cruz, sc393905; dilution 1:2000) or RUVBL2 antibodies (Santa cruz, sc374135, dilution 1:2000), and RPAP3 (ABclonal, A15239; dilution 1:2000).

Techniques: Expressing, ChIP-sequencing, RNA Sequencing, Two Tailed Test, Immunofluorescence

Journal: Nature Communications

Article Title: The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors

doi: 10.1038/s41467-022-33433-3

Figure Lengend Snippet: a The list of transcription factor motifs that were enriched at the RUVBL1- and RUVBL2-overlapping peaks and the corresponding p -values (which are calculated using HOMER with cumulative binomial distributions) is shown on the right. The transcription factors in blue were previously reported to function with RUVBL1 or RUVBL2. b Cluster analysis with RUVBL1, RUVBL2, Pol II, C-MYC, E2F1, P53, CTNNB1, and OCT4 in RUVBL1/2-overlapping peaks. c Distribution pie charts showing the percentage of the corresponding transcription factor peaks in each category (K1 to K6) shown in Fig. b . d The heatmap illustrating RUVBL2 globally colocalization with various transcription factors in MCF-7 cells. e Metagene analyses of RUVBL2 ChIP-Seq data for the regions of genes directly targeted by RUVBL2, all genes and posttranscriptional early response genes. f The cancer hallmark genes directly targeted by RUVBL2 (red) and the posttranscriptional early response genes after RUVBL2 depletion (black). g Snapshots showing RUVBL2 ChIP-Seq signals at C-MYC loci in GM12878, K562, U2OS, HeLa, MCF-7, HCT116 and HepG2 cells. h The survival probability changes over time for patients with breast cancer and high (red line) or low (black line) expression of RUVBL1 (upper panel) and RUVBL2 (bottom panel) as determined by the curve constructed with the Kaplan–Meier plotter. Significance was calculated by log-rank method. HR indicates the hazard ratio, and the confidence interval is shown in brackets. i Model of RUVBL2-activated transcription of genes with diverse cellular functions and Pol II clustering. RUVBL1/2 were shown to function as global transcription coactivators with various transcription factors and to directly promote Pol II cluster formation mediated by the RPB1 CTD.

Article Snippet: The IPed samples were analyzed with RPB1-CTD (Abcam, ab26721, dilution 1:3000) antibodies (to confirm the Pol II degradation), RPB1-NTD antibodies (dilution 1:3000) and RUVBL1 (Santa cruz, sc393905; dilution 1:2000) or RUVBL2 antibodies (Santa cruz, sc374135, dilution 1:2000), and RPAP3 (ABclonal, A15239; dilution 1:2000).

Techniques: ChIP-sequencing, Expressing, Construct

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: Preliminary Research of Main Components of Dll4/ Notch-VEGF Signaling Pathway Under High-Glucose Stimulation in vitro

doi: 10.2147/DMSO.S355004

Figure Lengend Snippet: Sequences of Primers for RT-PCR

Article Snippet: After the proteins were transferred on nitrocellulose membranes, antibodies specific for Dll4 (1:200, SC365429, Santa Cruz, CA), Notch1 (1:200, SC376403, Santa Cruz, CA), Notch4 (1:200, SC393893, Santa Cruz, CA) and the VEGF (1:200, SC365578, Santa Cruz, CA) were used for incubation.

Techniques:

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: Preliminary Research of Main Components of Dll4/ Notch-VEGF Signaling Pathway Under High-Glucose Stimulation in vitro

doi: 10.2147/DMSO.S355004

Figure Lengend Snippet: Cultured cells from control group, HG group and DAPT+HG group were harvested for mRNA and protein analyse. ( A ) RT-PCR showed the increased gene expression of Dll4, Notch1 and VEGF induced under HG stimulation, and Notch4 was expressed lower than that in control group. With DAPT interference, the results were reversed.The relative gene expression was normalized to β-actin. ( B and C ) The proteins from cells as described in A were collected for Western blot assay. Proteins expression showed the same results as the indicated gene expression in ( A ). Compared with control group, *P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After the proteins were transferred on nitrocellulose membranes, antibodies specific for Dll4 (1:200, SC365429, Santa Cruz, CA), Notch1 (1:200, SC376403, Santa Cruz, CA), Notch4 (1:200, SC393893, Santa Cruz, CA) and the VEGF (1:200, SC365578, Santa Cruz, CA) were used for incubation.

Techniques: Cell Culture, Control, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Western Blot, Expressing

Journal: Cell

Article Title: Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps

doi: 10.1016/j.cell.2021.11.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD20, D-10 clone, A647 dye, antibody , Santa Cruz , Catalog: sc-393894 AF647.

Techniques: Plasmid Preparation, Software

Journal: International Journal of Molecular Sciences

Article Title: B Cell Activating Factor (BAFF) Is Required for the Development of Intra-Renal Tertiary Lymphoid Organs in Experimental Kidney Transplantation in Rats

doi: 10.3390/ijms21218045

Figure Lengend Snippet: Intra-renal infiltrates, their microanatomical localization, and content of T and B lymphocytes. ( A ) shows intra-renal infiltrate expansion, which was measured using Histoquest software and was expressed as the cumulative area of infiltrates/area of the renal cortex. ( B ) shows the microanatomical localization of infiltrates, which was recorded as perivascular, periglomerular, or interstitial. ( C ) shows the intra-renal content of CD3 + T cells and CD20 + B cells, which was determined using Histoquest software after immunohistochemical staining and normalized to the area of renal cortex. ( D ) shows the ratio of intra-renal B/T cells in arbitrary units (AU). NR, no rejection (black); CR, chronic rejection (pink); CR + AB, chronic rejection and anti-BAFF antibody (green). Data is shown as individual data points per rat and group means. Statistical significance is shown as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: B cells were stained with mouse anti-rat CD20 antibody (Santa Cruz, sc-393894, Heidelberg, Germany), goat anti-mouse-biotin (Thermofisher 31804), and Strep-Cy3 (Dianova, 016-160-084, Hamburg, Germany).

Techniques: Software, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: B Cell Activating Factor (BAFF) Is Required for the Development of Intra-Renal Tertiary Lymphoid Organs in Experimental Kidney Transplantation in Rats

doi: 10.3390/ijms21218045

Figure Lengend Snippet: Effect of anti-BAFF treatment on intra-renal T and B cell zones and TLO formation. ( A ) shows representative allograft sections stained for CD3 (T cells, pink), CD20 (B cells, yellow), and Ki67 (proliferating cells, green) with distinct T and B cell zones. ( B ) shows the frequency of T cell (CD3 + ) and B cell (CD20 + ) zones, which were defined as dense clusters of predominantly one cell type. ( C ) shows the frequency of TLOs, which were defined as dense intra-renal infiltrates containing a T and a B cell zone. NR, no rejection (black); CR, chronic rejection (pink); CR + AB, chronic rejection and anti-BAFF antibody (green). Data is shown as group means and individual data points per rat. Statistical significance is shown as *** p < 0.001.

Article Snippet: B cells were stained with mouse anti-rat CD20 antibody (Santa Cruz, sc-393894, Heidelberg, Germany), goat anti-mouse-biotin (Thermofisher 31804), and Strep-Cy3 (Dianova, 016-160-084, Hamburg, Germany).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: B Cell Activating Factor (BAFF) Is Required for the Development of Intra-Renal Tertiary Lymphoid Organs in Experimental Kidney Transplantation in Rats

doi: 10.3390/ijms21218045

Figure Lengend Snippet: Intra-renal T and B cell proliferation and germinal center (GC) formation within TLOs. ( A ) shows the intra-renal content of proliferating Ki67 + CD3 + T cells and ( B ) proliferating Ki67 + CD20 + B cells, which were quantified using Histoquest software after immunohistochemical staining and normalized to the area of renal cortex. ( C ) shows a TLO with GC formation, which was defined as a dense cluster of Ki67 + proliferating cells (green) within a CD20 + B cell zone (yellow); the kidney section was from the CR group. NR, no rejection (black); CR, chronic rejection (pink); CR + AB, chronic rejection and anti-BAFF antibody (green). Data is shown as group means and individual data points per rat. Statistical significance is shown as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: B cells were stained with mouse anti-rat CD20 antibody (Santa Cruz, sc-393894, Heidelberg, Germany), goat anti-mouse-biotin (Thermofisher 31804), and Strep-Cy3 (Dianova, 016-160-084, Hamburg, Germany).

Techniques: Software, Immunohistochemical staining, Staining

Journal: JCI Insight

Article Title: Contribution of plasma cells and B cells to hidradenitis suppurativa pathogenesis

doi: 10.1172/jci.insight.139930

Figure Lengend Snippet: Box-and-whisker plots of BCR CDR3 expressions ( A ). The y axis shows normalized log-transformed BCR CDR3 expression. The x axis represents patient group. In all cases there were more BCR CDR3 sequences detected in HS skin compared with control healthy skin. Box-and-whisker plots of BCR gene segment expression. The y axis shows normalized log-transformed BCR gene segment expression. The x axis represents patient group. The Shannon diversity index for BCR CDR3 gene segment is plotted on the y axis. The x axis represents patient group. HS skin had a significantly more diverse BCR repertoire ( B ). Beta diversity–based principal coordinates analysis (PCoA) of BCR CDR3 sequences. Sample matrix was generated using Jaccard dissimilarities, and respective profiles were compared by PCoA. Each color represents 1 patient group, HS (red) and control (blue). This analysis revealed clear separation for κ and λ light chains but not Ig heavy chain ( C ) Hierarchical clustering of expressed TCR V/J gene segment expression. Heatmaps by clonal abundance across sample sets. Note good separation of HS from controls based upon clonal abundances in BCR κ and λ repertoires. Components of the complement pathway (C1q) and breakdown products of activated complement components (C3b, C4d) were increased in HS skin, particularly in the deeper layers of the skin ( n = 3) (scale bar: 100 μm) ( D ). Complement receptors, CR1 and CR2, were increased in the deeper layers of HS, along with IgG1 immune complex deposition ( n = 3) (scale bar: 100 μm) ( E ). Immunofluorescence of B cells (CD20) and plasma cells (CD138) showed primary localization of TNF to the plasma cell population in HS skin ( n = 3) (scale bar: 50 μm) ( F ). For A and B , the bold vertical line represents the median, and the upper and lower limits of the box represent the interquartile range (IQR). The whiskers represent 1.5× IQR.

Article Snippet: Overnight coincubation (4°C) was then performed using anti–human TNF (Abcam, catalog ab6671), anti–human CD20 (Santa Cruz Biotechnology, catalog sc-393894), and anti–human CD138 (LifeSpan Biosciences, LS-B9360-50).

Techniques: Whisker Assay, Transformation Assay, Expressing, Control, Generated, Immunofluorescence, Clinical Proteomics

Journal: JCI Insight

Article Title: Contribution of plasma cells and B cells to hidradenitis suppurativa pathogenesis

doi: 10.1172/jci.insight.139930

Figure Lengend Snippet: Analysis of the signal transduction networks using literature-based networks (Genomatix-Pathway System, GePS) demonstrated enrichment for pathways involved in B cell signaling and activation ( A ). To confirm the nature of the inflammatory infiltrate in HS and the localization of components of the enriched signaling pathways, we performed IHC in an excisional biopsy for CD3, CD20, and CD138. Plasma cells were the predominant inflammatory infiltrate and most prominent in the deeper layers of the skin surrounding a deeper sinus tract ( A ), accompanied by increased expression of BTK, SYK, and LCK ( B ) ( n = 3). Activation of key components of this signaling pathway was confirmed by IHC for both phospho-BTK and phospho-SYK ( n = 3) ( C ).

Article Snippet: Overnight coincubation (4°C) was then performed using anti–human TNF (Abcam, catalog ab6671), anti–human CD20 (Santa Cruz Biotechnology, catalog sc-393894), and anti–human CD138 (LifeSpan Biosciences, LS-B9360-50).

Techniques: Transduction, Activation Assay, Protein-Protein interactions, Clinical Proteomics, Expressing