Anti hdac2

Cells were lysed in protein extraction buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na₂P₂O₇, 1 mM Na₃VO₄, 5 μg/mL aprotinin, 5 μg/mL leupeptin, 1 mM PMSF, and protease inhibitor cocktail). Lysates containing equal amounts of proteins were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The blots were blocked with a 5% skim milk solution and incubated with the following antibodies: anti-Dicer, anti-cyclin A, anti-cyclin D1, anti-cyclin E, anti-p21^WAF1/CIP1, anti-EZH2,(Cell Signaling Technology, Danvers, MA), anti-HDAC2, anti-GAPDH and anti-CDK2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-N-cadherin, anti-E-cadherin, anti-vimentin and anti-fibronectin (BD Transduction, San Jose, CA), anti-DNMT1 (Abcam, Cambridge, MA) and anti-H3K27me3 (Millipore, Billerica, MA). The Immobilon™ Western blot detection system (Millipore, Billerica MA) was used to detect bound antibodies. The intensities of the Western blot bands were quantified using LAS 3000 (Fuji Photo Film Co., Japan).

Myc-CREPT, HA–HDAC1, HA–HDAC2, HA–HDAC3, HA–TCF4, Flag–β-catenin, and SuperTop-luciferase were constructed in this laboratory. Anti-HDAC2 (Santa Cruz, Santa Cruz, CA, USA, H54), anti-HA (Santa Cruz, F-7), anti-Myc (Santa Cruz, 9E10), anti-HDAC1 (CST, Danvers, MA, USA, 34589, 5356; Abcam, Cambridge, UK, ab19845), anti-HDAC3 (CST, 3949; Abcam, ab137704), anti-TCF4 (CST, 2569), anti-H3K27ac (Abcam, ab4729), anti-actin (Sigma, St. Louis, MI, USA, AC-15), anti-Flag (Sigma, M2), and anti-β-catenin (BD, Franklin Lakes, NJ, USA, 4171778) were used. We generated an anti-CREPT antibody in our laboratory [34 (link)]. Short interfering RNAs (siRNAs) against HDAC1 (siHDAC1-1: 5′-GCCGGUCAUGUCCAAAGUATT-3′; siHDAC1-2: 5′-GCGACUGUUUGAGAACCUUTT-3′) were used. The guider RNAs sequences for deleting CREPT were 5′-CGGTGCCACACGGAGACGAT-3′ and 5′-GCTAAGCCCCCTGTGACGTT-3′.

Carfilzomib was obtained from Selleck Chemicals. Pan-histone deacetylase (HDAC) inhibitors panobinostat (PS) and vorinostat (VS) were obtained from Novartis Pharmaceuticals (East Hanover, NJ) and Selleck Chemicals, respectively. All drugs were prepared as 10 mM stocks in 100% DMSO and stored in small aliquots at −80°C to prevent multiple free thaw cycles. Anti-phosphorylated (p)-ATR (S428), anti-p-CHK1(S345), anti-α/β tubulin, anti-DNA-PKcs, anti-acetyl histone H3 (K9/K14), anti-histone H3, anti-RAD52, anti-BRCA2 and anti-acetyl lysine antibodies were purchased from Cell Signaling Technology (Berverly, MA). Anti-hsp90α and anti-hsp70 antibody was purchased from Enzo Biosciences (Plymouth Meeting, PA). Anti-BRCA1 and anti-γ-H2AX antibodies were obtained from Millipore (Billerica, MA). Anti-CHK1, anti-ATR, anti-HDAC1, anti-HDAC2, and anti-HDAC3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin, anti-FLAG, anti-acetylated α-tubulin and anti-GFP antibodies and short hairpin RNAs against HDAC1, HDAC2 and HDAC3 were purchased from Sigma-Aldrich (St. Louis, MO). Acetylated-K69 hsp90 (Ac-K69 hsp90) antibody was previously described [24 (link)]. Anti-c-RAF antibody was purchased from BD Transduction Labs (San Jose, CA).

Nuclear protein was extracted and quantified using the BCA Protein Assay Kit (Life Technologies). Protein samples (10–20 µg) were electrophoresed in 4–12% Bis–Tris gels and transferred overnight to a nitrocellulose membrane at 4 °C. Primary antibodies used were anti-HDAC2 (Santa Cruz) and anti-CHD7 (#6505, Cell Signaling). Secondary antibodies were goat anti-rabbit IgG HRP (#31461, Thermo Fisher) and rabbit anti-goat IgG HRP (#31403, Thermo Fisher).

For the ChIP assay, NPCs derived from wild-type or Smek1/2 dKO E11.5 forebrain or transfected with 4 μg pUltra-hot-Mbd3-flag or a pUltra-hot-empty vector for Mbd3 gain-of-function experiments and with pLKO3G-shMbd3 or pLKO3G-shScramble for Mbd3 loss-of-function were treated with 1% formaldehyde for 10 min at room temperature and quenched with 0.125 M glycine for ten more minutes at room temperature. Cross-linked chromatin was sonicated to fragment DNA to 200–1,000 base pairs, and then immunoprecipitation was performed with rabbit anti-IgG, anti-Smek1 (Sigma), anti-Mbd3 (Cell Signaling), anti-HDAC1, anti-HDAC2, anti-MTA1, and acetyl histone H3 (Santa Cruz Biotechnology) antibodies overnight at 4°C, followed by incubation with 50 μl of magnetic Protein A/G Dynabeads (EMD Millipore). Abundance of sequences in immunoprecipitates was determined by PCR and normalized as a fold-value relative to input chromatin. Smek ChIP-seq data were analyzed with the MACS online tool, and cis-regulatory sequences were analyzed using the Genomic Regions Enrichment of Annotations Tool (GREAT) interface (http://bejerano.stanford.edu/great/public/html/). We also utilized the Intergrative Genomics Viewer (IGV v2.3) to visualize distribution of ChIP-seq–identified peaks in different genomic regions. Primer sets for ChIP-qPCR are listed in S6 Table.

The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and incubated on ice for 30 min with SDS lysis buffer. Equal amounts of cell lysates were separated by SDS-PAGE. Primary antibodies, including anti-Oct4 (Santa Cruz Biotechnology), anti-Nanog (Abcam), anti-HDAC1 (Sigma), anti-HDAC3 (Cell Signaling), anti-HDAC2 (Santa Cruz Biotechnology), anti-HDAC8 (Santa Cruz Biotechnology), anti-H4/acetyl-H4 (Millipore) anti-H3/acetyl-H3 (Millipore), anti-T/Bry (abcam), anti-Gata4 (Santa Cruz Biotechnology), anti-SMA (Sigma) and anti-GAPDH (Sigma) were used in this study. GAPDH was used as loading controls. After incubation with the appropriate secondary antibodies, signals were visualized by enhanced chemiluminescence (ECL) (ImageQuant LAS 4000 mini).