Hdac2

Western blot was done as previously described21 (link). Antibodies specific for the following targets were from Santa Cruz (Heidelberg, Germany): BAX, β-catenin, HSP90, histone deacetylase (HDAC)2, PIG3, p53 and p21. Anti-survivin was obtained from Novus Biologicals (Cambridge, UK).

Human prostate LNCaP and Du145 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI supplemented with 10% fetal bovine serum (FBS; Gibco-BRL, MD) and 1% antibiotic-antimycotic solution in a humidified 5% CO₂ atmosphere at 37°C. Antibodies against cleaved caspase-3, caspase-7, caspase-8, caspase-9, PARP, Survivin, XIAP, cIAP-2, BID, BAX, DR5, BCL-2 and acetylated p53 (Ac-p53) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-HDAC1, HDAC2, HDAC3, HDAC8, FLAG, p53, and actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-FLAG and β-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO). The anti-mouse or anti-rabbit secondary antibodies were purchased from Pierce (Rockford, IL). Delphinidin and TRAIL were obtained from Sigma-Aldrich (Fig. 1A).

Immunohistochemical staining was carried out as described in [25 (link)]. Primary antibodies used were as follows: HDAC2 (dilution 1:250, Santa Cruz Biotechnology, Dallas, TX, USA; #sc-55541), CIITA (dilution 1:300, Abcam, Cambridge, UK; #ab117598), and B2M (dilution 1:250, Cell Signaling, Danvers, MA, USA; #9899).

Immunoblotting was performed using standard protocols using c-MYC, p21, Ac-H3 (Lys9/Lys14), Ac-H4 (Lys5), p-MEK, MEK, p-ERK1/2, ERK1/2 (Cell Signaling, Danvers, MA, USA), BAX, HDAC1, HDAC2, HDAC3 (Santa Cruz Inc., Dallas, TX, USA), BRD4 (Bethyl Laboratories Inc., Montgomery, TX, USA), beta-actin (Sigma-Aldrich), USP17L5 (Abcam) primary antibodies and horse radish peroxidase (HRP) conjugated anti-mouse IgG or anti-rabbit IgG (GE Healthcare, Piscataway, NJ, USA) secondary antibodies.

For immunoblotting, whole cell protein extracts were prepared with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 10-% NP-40, 1% Triton-X and protease inhibitors), subjected to SDS-PAGE (10 or 12%) and blotted onto PVDF membranes (Roth, Karlsruhe, Germany). Primary antibodies against PARP, CDK4, CDK1, cyclin B1, cyclin E, p21, HDAC1, HDAC2, HDAC3, HDAC8, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, US). Anti-p27^Kip1, -cyclin D1, -CDK2, -phosphoCDK2 (Thr160), -phosphoCDK4 (Thr172), and -phosphoH2AX (Ser139) were purchased from Cell Signaling Technology (Danvers, MA, US), and anti-p53 from Dako (Glostrup, Denmark). Blots were developed using corresponding horseradish peroxidase- conjugated secondary antibodies (Dako, Jena, Germany) at room temperature for 1 h and the Amersham™ ECL™ prime western blotting detection reagent (GE Healthcare) in accordance with the manufacturer‘s protocol. Chemiluminescence signals were detected by the ChemiDocTouch Imaging System (BioRad Laboratories Inc., Herkules, CA) and images were processed using ImageLab 5.2 Software (BioRad Laboratories Inc.), normalized to their loading controls and expressed as fold-change (Δ) compared to controls.

The antibodies specific for HDAC1, HDAC2, HDAC3, HDAC8, Cip1/Waf1/p21, p16, CDK2, CDK4, β-Actin, histone H3, and secondary antibodies horseradish peroxidase-linked anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for cyclin D1, cyclin D2, cyclin E, CDK6 and p53 were obtained from Cell Signaling Technology, Inc. (Denver, MA). MG132, Valproic acid (VPA) and other chemicals of analytical grade were purchased from Sigma-Aldrich Corp. (St. Louis, MO).

MCF-7, SKBR-3, MCF-7/T7-MTA1, MEF WT, MEF MTA1^−/−, HCT-116 cells were used in the study. RPMI 1640 (Invitrogen, CA) supplemented with 10% FBS and 1X antibiotic/antimycotic (15240062, Gibco, CA) was used to maintain the cells in a humidified 5% CO₂ at 37 °C. Antibodies used were MTA1 (#5447, Cell Signaling Technology), DNMT3a (ab188470, Abcam), IGFBP3 (sc-9028, Santa Cruz), HDAC1 (ab46985, Abcam), HDAC-2 (sc-7899, Santa Cruz), Vinculin (sc-73614, Santa Cruz), RNA Polymerase II (A300-654A, Bethyl labs), DNMT3a (sc-20703, Santa Cruz), YY1 (#2185, Cell Signaling Technology) and β-Actin (sc-47778, Santa Cruz). Secondary antibodies conjugated to Horseradish peroxidase were obtained from Sigma-Aldrich. Chemiluminescence detection reagents were obtained from Millipore (WBKLS0100). All other reagents were purchased from Sigma-Aldrich, if not mentioned.