Journal: Communications Biology

Article Title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

doi: 10.1038/s42003-025-08413-0

Figure Lengend Snippet: a Differentially expressed TE subfamilies between GP5d and p53-KO GP5d cells. Scatter plot shows normalized RNA-seq read counts for TE subfamilies. Differentially expressed TE subfamilies are labeled by TE class. b Distinct TE subfamilies derepressed by CMEi in GP5d p53-KO cells. Expression changes for TE subfamilies (log2FC) were compared between different CME treatments in GP5d p53-KO cells. Significance symbols: **** indicates p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = non-significant |log2FC| < 1.5 or p > 0.05. c Loss of p53 is associated with stronger derepression of TEs by DNMTi-HDACi. Bar plots compare (i) the number of derepressed TE loci by DNMTi-HDACi in WT GP5d and two independent p53-KO clones and (ii) the number of derepressed TEs loci between LNCAP-1F5 p53-WT and p53-KO cells. d Rescue experiments showing that p53 reintroduction is associated with weaker derepression of TEs at the subfamily and locus levels by DNMTi-HDACi. Bar plot comparing the number of derepressed TE subfamilies and TE loci by DNMTi-HDACi in GP5d, GP5d p53-KO clones #1 and #2 and p53-transfected GP5d p53-KO clone #1. e Volcano plots showing the differentially expressed individual TE loci by DNMTi-HDACi in GP5d, OE19, and GP5d p53- KO cells. f Bar plot comparing p53 expression in GP5d and OE19 cells treated with DNMTi-HDACi vs. DMSO control (unpaired two-sided t-test). g Genome browser snapshot of the TP53 gene locus showing the ChIP-seq signals for H3K27ac and H3K4me3 and an RNA-seq signal track for both DMSO control and DNMTi-HDACi-treated GP5d cells. h Comparison of TE expression changes induced by DNMTi-HDACi treatment between TEs harboring p53REs with different strengths. Expressed TEs with p53REs were stratified into five grades from least to most likely p53REs with transactivation potential using p53retriever . Boxplots show the number of up- and downregulated TE loci upon DNMTi-HDACi for each grade, significantly differentially expressed loci are marked with red (Adjusted p < 0.05, |log2FC| > 1). Comparisons between grades were performed with one-sided Wilcoxon tests. Number of LINEs and LTRs loci for each grade are shown in Supplementary Data . Source data are provided as Supplementary Data .

Article Snippet: Next day cells were transfected with p53 expressing pIRES2-EGFP-p53 WT plasmid (addgene # 49242).

Techniques: RNA Sequencing, Labeling, Expressing, Clone Assay, Transfection, Control, ChIP-sequencing, Comparison

Journal: Communications Biology

Article Title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

doi: 10.1038/s42003-025-08413-0

Figure Lengend Snippet: a TF motif enrichment at TE sequences derepressed by DNMTi-HDACi in GP5d and OE19 cells. After performing motif enrichment analysis for individual motifs, similar motifs were combined into motif clusters from ref. . The representative TF clusters are labeled on the right. The minimum E-value found for an individual TF for each motif cluster was plotted in the final figure. b Boxplots comparing the expression of derepressed LTR12C in DMSO and DNMTi-HDACi GP5d, OE19 and GP5d-p53-KO cells (two-sided Wilcoxon paired test). The lower and upper hinges of the boxes represent the 25th to 75th percentiles, the midline is the median, and the whiskers extend from the hinges to the minimum and maximum values by 1.5 * interquartile range (IQR). c , Venn diagram showing the overlap between LTR12Cs derepressed by DNMTi-HDACi in GP5d and OE19 (shown in Fig. 4b). d Metaplots of ATAC-seq and CUT&TAG for H3K27me3, H3K4me1, RNAPII and Ser5p-RNAPII at derepressed LTR12Cs in DNMTi-HDACi OE19 cells (shown in Fig. 4b). Derepressed LTR12C elements show a poised chromatin state in OE19 cells, whereas LTR12C in GP5d cells were enriched with repressive H3K27me3 marks (see Supplementary Fig. ). e Heatmap showing the KAS-seq signal at derepressed LTR12C elements in DNMTi-HDACi OE19 cells (shown in Fig. 4b). f Volcano plot of nanopore sequencing data comparing CpG methylation levels at derepressed LTR12C elements (n = 499) in control and DNMTi-HDACi GP5d cells (shown in Fig. 4b). Significance was determined with a one-sided Fisher’s exact test. P -values were corrected with Benjamini-Hochberg method. g Heatmaps showing the ChIP-seq signals for H3K27ac and H3K4me3 in control and DNMTi-HDACi GP5d cells (shown in Fig. 4b). h Heatmaps showing the ChIP-seq signals for H3K27ac and H3K4me3 in control and DNMTi-HDACi OE19 cells (shown in Fig. 4b). i Derepressed LTR12C elements in DNMTi-HDACi GP5d and OE19 cells. Volcano plots show the changes in gene expression for derepressed LTR12C-associated genes for OE19 cells. Analysis of differentially expressed genes in OE19 cells with DNMTi-HDACi revealed significant upregulation of genes in the vicinity of derepressed LTR12C elements (±50 kb). In total, 102 out of 132 and 70 out of 89 differentially expressed genes were upregulated in GP5d and OE19 cells, respectively. j Genome browser snapshot of a derepressed LTR12C in control and DNMTi-HDACi GP5d cells. Panels show the ChIP-seq signals for H3K27ac, H3K4me3, and an RNA-seq signal track. k Genome browser snapshot of a derepressed LTR12C in control and DNMTi-HDACi OE19 cells. Each panel shows the ChIP-seq signals for H3K27ac, H3K4me3 and an RNA-seq signal track. Source data are provided as Supplementary Data .

Article Snippet: Next day cells were transfected with p53 expressing pIRES2-EGFP-p53 WT plasmid (addgene # 49242).

Techniques: Labeling, Expressing, Nanopore Sequencing, CpG Methylation Assay, Control, ChIP-sequencing, Gene Expression, RNA Sequencing

Journal: Communications Biology

Article Title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

doi: 10.1038/s42003-025-08413-0

Figure Lengend Snippet: a Comparison of SETDB1 KO/inhibition-induced changes in the expression of TE subfamilies between six different cell lines. Differential expression analysis of RNA-seq data was performed with DESeq2. All TE subfamilies with absolute log2FC > 1.5 (treatment vs DMSO or A375 KO vs A375) and adjusted p < 0.05 in at least one CMEi treatment in one cell line were selected and their expression changes in log2FC was plotted. Rows and columns are clustered with hierarchical clustering. b Number of differentially expressed TE loci by SETDB1 KO/inhibition in A375, WT and p53-KO GP5d, OE19, and WT and p53-KO LNCaP-1F5 cells. c The proportion of TEs derepressed by SETDB1 KO/inhibition belonging to the major TE classes among the derepressed TEs by in A375, WT and p53-KO GP5d, OE19, and WT and p53-KO LNCaP-1F5 cells. d Number of Alu SINEs derepressed by SETDB1 KO/inhibition in A375, WT and p53-KO GP5d, OE19, and WT and p53-KO LNCaP-1F5 cells. Number above the bar represents the percentage of Alu SINEs in total derepressed TEs. e Motif enrichment analysis for TEs derepressed by SETDB1 KO/inhibition in A375, WT and p53-KO GP5d, OE19, and WT and p53-KO LNCaP-1F5 cells. The analysis was performed as described in Fig. . Source data are provided as Supplementary Data .

Article Snippet: Next day cells were transfected with p53 expressing pIRES2-EGFP-p53 WT plasmid (addgene # 49242).

Techniques: Comparison, Inhibition, Expressing, Quantitative Proteomics, RNA Sequencing

Journal: Communications Biology

Article Title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

doi: 10.1038/s42003-025-08413-0

Figure Lengend Snippet: a Boxplots showing a comparison of expression of transcriptionally active IR-Alu SINEs (total sum of RNA-seq reads for DMSO and DNMTi-HDACi ≥ 5, see “Methods” for details) in DMSO and DNMTi-HDACi GP5d, OE19, and GP5d p53-KO cells. Majority of IR-Alu SINEs are derepressed by DNMTi-HDACi (two-sided Wilcoxon paired test). The lower and upper hinges of the boxes represent the 25th to 75th percentiles, the midline is the median, and the whiskers extend from the hinges to the minimum and maximum values by 1.5 * IQR. b Heatmap showing the ChIP-seq signal for H3K27ac at IR-Alu SINEs in DMSO and DNMTi-HDACi GP5d and OE19 cells. c Comparison of normalized RNA-seq read counts for ADAR1 gene in DMSO and DNMTi-HDACi GP5d, OE19, and GP5d p53-KO cells. The graph shows mean ± SD values for three biological replicates (two-sided unpaired t-test). d Microscopy images for GP5d cells treated with DMSO or DNMTi-HDACi. DNA was stained with DAPI (blue), and dsRNA was stained using the J2 antibody (green). All scale bars are 20 μm. Cytoplasmic levels of dsRNA increased in DNMTi-HDACi treated GP5d cells as compared to the DMSO. e Microscopy images for dsRNA staining in OE19 cells treated with DMSO or DNMTi-HDACi, as shown in Fig. 6d. f Microscopy images for dsRNA staining in GP5d p53-KO cells treated with DMSO or DNMTi-HDACi, as shown in Fig. 6d. g Alu editing index (AEI) was calculated by using RNAeditingIndexer tool on RNA-seq data. Bar plots showing AEI for DMSO and DNMTi-HDACi GP5d, OE19, and GP5d p53-KO cells. The graph shows mean ± SD values for three biological replicates (two-sided unpaired t-test). h Bar plots comparing RIG1 gene expression in DMSO and DNMTi-HDACi GP5d, OE19, and GP5d p53-KO cells. The graph shows mean ± SD values for three biological replicates (two-sided unpaired t-test). i qRT-PCR data showing RIG-I mRNA expression in GP5d and OE19 cells treated with DMSO and DNMTi-HDACi (GAPDH normalized). The graph shows mean ± SD values for three biological replicates. Source data are provided as Supplementary Data .

Article Snippet: Next day cells were transfected with p53 expressing pIRES2-EGFP-p53 WT plasmid (addgene # 49242).

Techniques: Comparison, Expressing, RNA Sequencing, ChIP-sequencing, Microscopy, Staining, Gene Expression, Quantitative RT-PCR

Journal: Communications Biology

Article Title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

doi: 10.1038/s42003-025-08413-0

Figure Lengend Snippet: a SETDB1i/KO increases the expression of TE-chimeric transcripts. Boxplots show the expression of TE-chimeric transcripts in A375, GP5d, OE19, and GP5d p53-KO cells with and without SETDB1i/KO. Expression of TE-chimeric transcripts was analyzed with the TEprof2 pipeline . P -values were calculated with a two-sided Wilcoxon test (n = 53, 24, 144, and 141 differentially expressed TE-chimeric transcripts in A375, GP5d, GP5d p53-KO, and OE19, respectively). The lower and upper hinges of the boxes represent the 25th to 75th percentiles, the midline is the median, and the whiskers extend from the hinges to the minimum and maximum values by 1.5 * IQR. b Analysis of TE subfamilies from which the TE-chimeric transcripts are derived from upon SETDB1i/KO. The counts for TE-chimeric transcripts are log2-transformed. c DNMTi-HDACi increases the expression of TE-chimeric transcripts. Boxplots show the expression of TE-chimeric transcripts in DMSO and DNMTi-HDACi GP5d, GP5d p53-KO, and OE19 cells. P -values were calculated with a two-sided Wilcoxon test (n = 35, 175, and 193 for GP5d, OE19, and GP5d p53-KO cells, respectively). The boxplot features are as in Fig. 7a. d Cell type-specific expression of TE subfamilies forming TE-chimeric transcripts by DNMTi-HDACi in cancer cell lines. The counts for TE-chimeric transcripts are log2-transformed. e ISGs are upregulated by DNMTi-HDACi. ISG log2FCs are plotted for DNMTi-HDACi treated GP5d, OE19 and GP5d p53-KO cells. f SETDB1i and DNMTi-HDACi increased levels of Serine 477 phosphorylated IRF7. Western blot compares Ser477-phospho-IRF7 and total IRF7 protein levels in GP5d, OE19 and GP5d p53-KO cells treated with different CME inhibitors. g qRT-PCR data showing IFNα mRNA expression in GP5d and OE19 cells treated with DMSO, DNMTi-HDACi, and SETDB1i (GAPDH normalized). ISGs are upregulated by DNMTi-HDACi and SETDB1i in GP5d and OE19 cells. The graph shows mean ± SD values for three biological replicates. Source data are provided as Supplementary Data .

Article Snippet: Next day cells were transfected with p53 expressing pIRES2-EGFP-p53 WT plasmid (addgene # 49242).

Techniques: Expressing, Derivative Assay, Transformation Assay, Western Blot, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Calcium transients regulate the apical emergence of basally located progenitors during Xenopus skin development

doi: 10.1038/s41467-025-61610-7

Figure Lengend Snippet: A Representative MIP image of the apical surface area of a MCC expressing calmodulin-GFP. Calmodulin displays colocalization with the basal bodies and the apical actin network. B Schematic depicting the localization of calmodulin in a MCC in relation to the basal bodies and the apical actin network. C Representative images of the skin epithelium from stage 24 control and calmodulin inhibitor-treated embryos. MCC fail to fully integrate into the superficial skin epithelium when calmodulin activity is blocked, as evident by their small apical surface area. D Quantification of the apical surface area of MCCs from control ( n = 100 MCCs) and calmodulin inhibitor-treated embryos ( n = 100 MCCs), 5 different embryos. Two-sided unpaired student’s t test; **** p < 0.0001; mean ± SEM. E Representative images of the skin neuroepithelium from embryos expressing wild-type calmodulin or Ca 2+ binding deficient calmodulin mutant (CALM1234). Expression of CALM1234 results in defective MCC apical emergence (green arrows) while expression of WT calmodulin does not affect MCC apical emergence (white arrows). F Quantification of MCC apical emergence upon expression of WT and Ca 2+ binding deficient mutant calmodulin. Two-sided χ 2 test; ** * * p < 0.0001. G Quantification of the apical surface area of MCCs expressing WT and Ca 2+ binding deficient mutant calmodulin. N = 80 control MCCs, 46 MCCs expressing WT calmodulin and 20 MCCs expressing mutant calmodulin from 4 embryos injected with WT calmodulin DNA and 4 embryos injected with mutant CALM1234 DNA. Two-sided unpaired student’s t test; **** p < 0.0001; mean ± SEM. Scale bars: A : 2 μm, D , F : 20 μm. Source data are provided as a Source data file.

Article Snippet: For microinjection of DNA constructs, atub:UtrGFP, atub:GECO-RED, PACR (Addgene #55774), atub:PACR, CALM-GFP (Addgene #47602), CALMWT (Addgene #111499), CALM1234 (Addgene #111518).

Techniques: Expressing, Control, Activity Assay, Binding Assay, Mutagenesis, Injection

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet:

Article Snippet: pCaM12(D20A/D56A)/pIRES2-eGFP , Addgene , Cat#111512.

Techniques: Control, Protease Inhibitor, Mutagenesis, DNA Purification, Recombinant, Software

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet:

Article Snippet: pCaM34(D93A/D129A)/pIRES2-eGFP , Addgene , Cat#111517.

Techniques: Control, Protease Inhibitor, Mutagenesis, DNA Purification, Recombinant, Software

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: RvD1 prevents LPS-induced SHP2 oxidation and its inactivation in the protection of AJ integrity. A. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in presence and absence of RvD1 (200 ng/ml) for 30 min and PTP activity was measured using PTP-specific phosphopeptide as a substrate. B & C. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in presence and absence of RvD1 (200 ng/ml) for the indicated time periods, cell extracts were prepared and equal amounts of protein from control and each treatment were immunoprecipitated with VE-cadherin or SHP2 antibodies and the immunocomplexes were analyzed by IB for the indicated proteins. D. All the conditions were same as in panel B except that after immunoprecipitation with SHP2 antibodies the immunocomplexes were assayed for SHP2 activity as described in panel A. E. Quiescent HUVECs that were treated with and without LPS (500 ng/ml) in the presence and absence of Allopurinol (50 μM) for 30 min were analyzed for SHP2 activity as described in panel D. F. All the conditions were same as in panel B except the cell extracts were immunoprecipitated with SHP2 antibodies and the immunocomplexes were immunoblotted for Cys sulphonate to measure SHP2 Cys oxidation and the blot was reprobed for total SHP2 levels. G. All the conditions were the same as in panel E except that the cell extracts were analyzed for SHP2 Cys oxidation as described in panel F and the blot was reprobed for total SHP2 levels. H. HUVECs were transiently transfected with empty vector (EV) or Myc-tagged recombinant SHP2 expression vector (WT and C459S mutant), grown to confluence, quiesced, treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and cell extracts were prepared. An equal amount of protein from control and each treatment was immunoprecipitated with Cysteine sulphonate antibody and the immunocomplexes were analyzed by IB for Myc to show SHP2 Cysteine oxidation. An equal amount of protein from control and each treatment was also analyzed by WB for Myc to show the overexpression of SHP2. I. HUVECs that were transfected with WT or mutant SHP2 expression vector and quiesced were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and analyzed for SHP2 activity as described in panel D.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Activity Assay, Phospho-proteomics, Control, Immunoprecipitation, Transfection, Plasmid Preparation, Recombinant, Expressing, Mutagenesis, Over Expression

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: Pharmacological inhibition of SHP2 blunts the capacity of RvD1 in the attenuation of LPS-induced Frk activation, α-catenin and VE-cadherin Tyr phosphorylation and AJ disruption. A. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with or without PHPS1 (10 μM), a potent inhibitor of SHP2, for 30 min, cell extracts were prepared and an equal amount of protein from control and each treatment was immunoprecipitated with pTyr or VE-cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins. The same cell extracts were also analyzed for the indicated protein total levels. B. All the conditions were the same as in panel A except that the quiescent HUVEC monolayer after the treatments was stained double immunofluorescently for α-catenin and VE-cadherin as described in Figure legend 2E. C & D. Quiescent HUVEC monolayer was treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with or without PHPS1 (10 μM) for 2 h or the indicated time periods and subjected to dextran flux (C) or TER (D) assays, respectively. The bar graphs represent Mean±SD values of three experiments. *, p<0.05 vs control; # , p<0.05 vs LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Disruption, Control, Immunoprecipitation, Staining

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: Both ALX/FPR2 and GPR32 mediate the protective effects of RvD1 on LPS-induced endothelial AJ disruption and its barrier dysfunction. A. Cell extracts of control and various time periods of LPS (500 ng/ml)-treated HUVECs were analyzed by WB for ALX/FPR2 and GPR32 levels using their specific antibodies. B-D. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) in combination with and without Boc2 (3 μM), ALX/FPR2 inhibitor, for 30 min and XO activity (B), ROS production (C) and SHP2 activity (D) were measured. E. All the conditions were the same as in panel B except that cell extracts were prepared, and equal amounts of protein from control and each treatment were immunoprecipitated with pTyr or VE-Cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same cell extracts were also analyzed by WB for the total levels of the indicated proteins. F & G. Quiescent HUVEC monolayer was treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with and without Boc2 (3 μM) for 2 h or the indicated time periods and subjected to dextran flux (F) and TER (G) assays, respectively. H-J. Quiescent HUVECs were incubated with either control IgG or GPR32 IgG (10 μg/ml) alone or in combination with and without Boc2 (3 μM) for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and XO activity (H), ROS production (I) and SHP2 activity (J) were measured. K. All the conditions were the same as in panel H except that cell extracts were prepared and equal amounts of protein from control and each treatment were immunoprecipitated with pTyr or VE-cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same cell extracts were also analyzed by WB for the total levels of the indicated proteins. L. The quiescent HUVEC monolayer that was incubated with either control IgG, GPR32 IgG (10 μg/ml), Boc2 (3 μM) alone or in combination for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min was stained double immunofluorescently for α-catenin and VE-cadherin as described in Figure legend 2E. M & N. Quiescent HUVECs monolayer was incubated with either control IgG or GPR32 IgG (10 μg/ml) alone or in combination with and without Boc2 (3 μM) for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 2 h or the indicated time periods and subjected to dextran flux (M) and TER (N) assays, respectively. The bar graphs represent Mean±SD values of three experiments. *, p<0.05 vs control or control IgG; # , p<0.05 vs LPS or con IgG+LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Disruption, Control, Activity Assay, Immunoprecipitation, Incubation, Staining

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: RvD1 attenuates LPS-induced aortic endothelial AJ disruption and hyper-permeability via blocking XO activity and SHP2 inactivation. A. C57BL/6 mice which were kept on chow diet were administered intraperitoneally with RvD1 (10 μg/kg body weight) every 2 days for 3 times before injecting LPS (5 mg/kg body weight) and 24 h later the aortas were isolated, tissue extracts were prepared and an equal amount of protein from each condition was analyzed for XO activity as described in Figure legend 4B. B. All the conditions were the same as in panel A except that tissue extracts containing an equal amount of protein from each condition were immunoprecipitated with Cys sulphonate antibodies and the immunocomplexes were analyzed by IB for SHP2. The same tissue extracts were analyzed by WB for total SHP2 levels. C. All the conditions were the same as in panel A except that tissue extracts were analyzed for SHP2 activity as described in Figure legend 5D. D & E. All the conditions were the same as in panel A except that tissue extracts were immunoprecipitated with pTyr (D) or VE-cadherin (E) antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same tissue extracts were analyzed by WB for the indicated protein total levels. F. All the conditions were same as in panel A except that after isolation the aortas were opened longitudinally, fixed, permeabilized, blocked and co-immunostained for α-catenin and VE-cadherin as described in Figure legend 2E. G. All the conditions were the same as in panel A except that mice were anesthetized and 0.1 ml of 1% Evans Blue (EB) dye was injected into the tail vein. After 20 min, the blood vessels were perfused with PBS through the left ventriculum and the aortas were isolated and photographed. After taking the pictures, the aortas were minced, incubated in formaldehyde solution at 55 °C for 24 h, centrifuged and the optical density of the supernatant was measured at 610 nm in SpectraMax 190 spectrophotometer (Molecular Devices). The aortic endothelial barrier permeability was expressed as ng of EB dye extravasated per mg aorta. The bar graphs represent Mean±SD values of three experiments with 2 animals/group or 5 animals minimum. *, p<0.05 vs control; # , p<0.05 vs LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Disruption, Permeability, Blocking Assay, Activity Assay, Isolation, Immunoprecipitation, Injection, Incubation, Spectrophotometry, Control