anti phospho hdac3 ser 424 rabbit polyclonal antibody  (Cell Signaling Technology Inc)

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 3. HDAC3 is required for extracellular matrix homeostasis and remodeling of semilunar valves. A, Movat’s pentachrome staining of remodeling aortic valve shows an increase in proteoglycans (arrows, blue) from E13.5 to E18.5 in Hdac3Isl1KO aortic valves with a reduction and remodeling of proteoglycans in control valves. B, Movat’s pentachrome staining of remodeling pulmonic valve shows an increase in proteoglycans (arrows, blue) from E13.5 to E18.5 in Hdac3Isl1KO pulmonic valves with a reduction and remodeling of proteoglycans in control valves. C, Masson’s trichrome staining demonstrates disorganized collagen expression (arrow, blue) in Hdac3Isl1KO E18.5 aortic valve. D, Masson’s trichrome staining demonstrates disorganized collagen expression (arrow, blue) in Hdac3Isl1KO E18.5 pulmonic valve. E, Verhoeff-Van Gieson (VVG) staining shows disorganized collagen expression (arrow, red) in Hdac3Isl1KO E18.5 aortic valve. F, Verhoeff-Van Gieson staining shows disorganized collagen expression (arrow, red) in Hdac3Isl1KO E18.5 aortic valve. G, Movat’s pentachrome-stained sections showincreasedproteoglycans(blue)inaorticvalvecuspsofE18.5Hdac3Mef2CKOhearts.H,Movat’spentachromestainingrevealsincreasedproteoglycans(blue) in E18.5 Hdac3Mef2CKO pulmonic valve. I, representative images of cleaved caspase-3 immunostaining in E13.5 semilunar valves. Arrows, positive staining. J, quantification of cleaved caspase-3-positive cells in E13.5 control and Hdac3Isl1KO aortic valves. K, quantification of cleaved caspase-3-positive cells in E13.5 control and Hdac3Isl1KO pulmonic valves. L, schematic model depicting disorganized extracellular matrix and reduced apoptosis in Hdac3Isl1KO E13.5 semilunar valves. M, schematic model showing hyperplastic, enlarged, and disorganized Hdac3Isl1KO E18.5 semilunar valves.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Staining, Control, Expressing, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 4. HDAC3 is a critical regulator of TGF- signaling pathway. A–E, IPA of microarray data from E9.5 Hdac3Isl1KO hearts. A, cardiovascular development and function subcategories significantly dysregulated in E9.5 Hdac3Isl1KO hearts, sorted by significance, from IPA diseases and function analysis. B, classes of congenital heart anomalies affected in E9.5 Hdac3Isl1KO hearts, sorted by significance, from IPA diseases and function analysis. C, 30 most significant upstream regulatorsfromIPAupstreamanalysisofE9.5Hdac3Isl1KOmicroarraydata.D,clusteredheatmapofdifferentiallyexpressedgenesrelatedtoupstreamregulator TGF-1 from IPA upstream analysis of Hdac3Isl1KO microarray data. E, top 30 upstream regulators, based on number of associated genes differentially expressed in E9.5 Hdac3Isl1KO hearts from IPA upstream analysis. F, transcripts for Sumo1, Nrp2, Smad4, Snai1, Tgf-1, and Kpnb1 were detected by real-time qPCR in Hdac3F/F and Hdac3Isl1KO outflow tract with right ventricle derived from E9.5 embryos (mean S.E. (error bars), n 3). G–J, ChIP-qPCR analysis of HDAC3 recruitment to promoter-proximal regions of TGF- pathway genes performed in wild-type E9.5 outflow tract (mean S.E., n 3). K and L, ELISA for TGF-1 (K) and phospho-SMAD2/3 (L) was performed in Hdac3F/F and Hdac3Isl1KO outflow tracts (mean S.E., n 3 (K) and n 4 (L)). M, phospho-SMAD2/3 immunostaining (arrows) in E9.5 Hdac3Isl1KO; R26R-LacZ/ hearts.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Microarray, Derivative Assay, ChIP-qPCR, Enzyme-linked Immunosorbent Assay, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 8. HDAC3 epigenetically silences TGF-1 within valvular mesenchymal cells by recruiting PRC2 complex to the NCOR complex. A, relative mRNA levels of TGF-1 in isolated cardiac endothelial cells (EC) or cardiac mesenchymal cells (MC) from E10.5 Hdac3F/F outflow tract cushion explants, infected either with CRE or GFP control lentivirus. B, relative mRNA levels of TGF-1 in isolated cardiac endothelial cells or dissected semilunar valves (SL) derived from Hdac3Isl1KO and Hdac3F/F E14.5 hearts. C, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in isolated cardiac endothelial cells or cardiac mesen- chymal cells from E10.5 outflow tract cushion explants. D, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in isolated cardiac endothelial cells or dissected semilunar valves from E14.5 hearts. E–K, ChIP-qPCR analysis of H3K27 trimethylation (E), H3K27 acetylation (F), RNA polymerase II (G), CREBBP (H), EZH2 (I), EED (J), and SUZ12 (K) upstream of TGF-1 in Hdac3Isl1KO and control E14.5 semilunar valves. L, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in E14.5 valvular mesenchymal cells infected with either control shRNA (sc-shRNA), EZH2 shRNA, or NCOR1 shRNA. M, ChIP-qPCR analysis of NCOR1 upstream of TGF-1 in Hdac3Isl1KO and control E14.5 semilunar valves. N, Co-ChIP for HDAC3 and either EZH2, NCOR1, H3K27me3, H3K27ac, or polymerase II upstream of TGF-1 in E14.5 wild-type dissected semilunar valves. O, total lysates from E14.5 wild-type pooled semilunar valves were immunoprecipitated (IP) by EZH2 antibody, and Western blot was performed using HDAC3 antibody. -Tubulin is shown as an input control. HDAC3 was quantified and normalized to total input -tubulin using ImageJ software (mean S.E. (error bars), n 3). P, ChIP-qPCR analysis of H3K27me3 upstream of TGF-1 in E14.5 valvular mesenchymal cells infected with either control shRNA, EZH2 shRNA, or NCOR1 shRNA.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Isolation, Infection, Control, Derivative Assay, ChIP-qPCR, shRNA, Immunoprecipitation, Western Blot, Software

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 9. HDAC3 functions in a deacetylase-independent manner to regulate EndMT and epigenetic silencing of TGF-1. A, HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression constructs were transfected in HEK-293T cells. Expression was detected by Western blot from whole cell lysates using FLAG antibody. GAPDH is shown as a loading control. B, HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression was quantified and normalized to total input GAPDH using ImageJ software (mean S.D. (error bars), n 3). C, HDAC activity of HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression was quantified against a pseudosubstrate. D, EndMT assay of control- or Cre-infected E10.5 Hdac3F/F outflow tract cushion explants co-infected with GFP, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses, imaged 24 h after isolation. E, quantification of average radial migration, measured in eight directions, of control- or Cre-infected E10.5 Hdac3F/F outflow tract cushion explants co-infected with GFP, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses, measured 24 h after isolation(mean S.E.(errorbars),n3).F,relativemRNAlevelsofTgf-1incontrol-orCre-infectedE14.5Hdac3F/Fvalvularmesenchymalcellsco-infectedwith control, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses (mean S.E., n 3). G, a 1309-bp TGF-1 promoter luciferase reporter (WT) or a truncated, 1267-bp TGF-1 promoter luciferase reporter, lacking an HDAC3-enriched region (mutant) were transfected in murine endothelial cells with and without an HDAC3-FLAG or HDAC3H134A,H135A-FLAG expression plasmid. Induction is represented as a ratio of firefly and Renilla luciferase activity. H–L, ChIP-qPCR analysis of H3K27 acetylation (H), H3K27 trimethylation (I), EZH2 (J), EED (K), and SUZ12 (L) upstream of TGF-1 in control- or Cre-infected E14.5 Hdac3F/F valvular mesenchymal cells co-infected with control, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses (mean S.E., n 3).

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Histone Deacetylase Assay, Expressing, Construct, Transfection, Western Blot, Control, Software, Activity Assay, Infection, Isolation, Migration, Luciferase, Mutagenesis, Plasmid Preparation, ChIP-qPCR

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 10. Summary of phenotypes and proposed model of HDAC3 function within second heart field progenitor cells and second heart field-derived mesenchymal cells. A, loss of HDAC3 in second heart field progenitor cells leads to outflow tract and semilunar valve pathologies. Strikingly, genetic deletion of HDAC3 in differentiated mesenchymal and smooth muscle cells (Hdac3TaglnKO) recapitulates the majority of these phenotypes. However, deletion of HDAC3 in differentiated cardiomyocytes (Hdac3Myh6KO) or endothelial cells (Hdac3Cdh5KO) did not recapitulate the cardiovascular defects observed in Hdac3Isl1KO embryos. B, Hdac3Isl1KO hearts exhibit disorganized collagen and elastin within dilated aortic walls and hyperplastic semilunar valves containing activated myofibroblasts and disorganized extracellular matrix. In both control and Hdac3Isl1KO cardiac endothelial cells, the upstream regulatory region of TGF-1 is occupied by RNA polymerase II and CREBBP and exhibits H3K27 acetylation concomitant with TGF-1 expression. In control semilunar valves, endothelial cells undergo EndMT to become mesen- chymalcells.Inthesemesenchymalcells,NCOR1,HDAC3,andPRC2complex(EZH2,EED,andSUZ12)arerecruitedtotheupstreamregulatoryregionofTGF-1,which becomes trimethylated on histone H3 Lys-27, and TGF-1 expression is epigenetically silenced. In Hdac3Isl1KO hearts, EZH2, EED, and SUZ12 are not recruited to the TGF-1 regulatory region, RNA polymerase II and CREBBP are present, and histone H3 Lys-27 remains acetylated, favoring aberrant expression of TGF-1 in mesen- chymal cells. TGF-1 activates mesenchymal cells to become myofibroblasts, which perpetuate EndMT and activation of mesenchymal cells through continued induction of TGF-1 and aberrant expression of extracellular matrix, including proteoglycans and collagen.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Derivative Assay, Control, Expressing, Activation Assay