Hdac8

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).

The human melanoma cell lines A375 and A2058 used in this study were obtained and grown as previously described [32 (link)]. The following reagents and primary antibodies were used: HDAC1, rabbit, Santa Cruz SC-7872; HDAC2, mouse, Santa Cruz SC-9959; HDAC3, rabbit active motif 40968 IB; HDAC4, rabbit, active motif 40969; HDAC5, mouse, Santa Cruz SC-133225; HDAC6, rabbit, Santa Cruz SC-11420; HDAC7, mouse, Santa Cruz SC-74563; HDAC8, mouse, Santa Cruz SC-17778; anti-HDAC9 antibody (ab18970), Abcam; anti-HDAC10 antibody (ab18971), Abcam; anti-HDAC11, ab18973, abcam; anti-ERK (tERK), anti-phospho-ERK (pERK), anti-AKT (tAKT), anti-phospho-AKT-T308 (pAKT-T308), anti-phospho-AKT-S473 (pAKT-S473), anti-caspase 3, anti-cleaved caspase 3, anti-caspase 8, anti-cleaved caspase 8, anticaspase 9, anti-cleaved caspase 9 (Cell Signaling Technology, Danvers, MA, USA), anti-EGFR (tEGFR), anti-Cathepsin-D, anti-VEGF, anti-MMP9, anti-E-cadherin, and anti-GAPDH (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Lipofectamine 2000 reagent was obtained from Invitrogen (Carlsbad, CA, USA, Cat. No 11668-019).

20 μg of total cell lysates were subjected to a 10% polyacrylamide sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and blots were transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes with the blots were then incubated with 5% non-fat milk in PBS with Tween-20 for 1 h to prevent non-specific binding before being incubated overnight at 4 °C in specific primary antibodies against HDAC1, HDAC7, HDAC8, E-cadherin, Vimentin, Ac-H3, Ac-H4, H3K4me2, H3K9me2 and Histone H3 (Santa Cruz Biotechnology, CA, USA) followed by incubation in peroxidase - conjugated secondary antibody at room temperature for 1 h, washed with PBST three times, then the protein signals were observed using the UVP BioSpectrum system (Analytic Jena Company).

Following the treatment under the indicated conditions, cell extracts were prepared with lysis buffer from cell signaling containing protease inhibitor. The lysates were centrifuged at 20,000× g for 20 min at 4 °C and used for Western blot analysis. Proteins were separated via SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were blocked for 30 min in 5% (w/v) non-fat skim milk in phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST). The blocked membranes were incubated with the indicated antibody for 2 h or overnight at 4 °C. After washing with 1× PBST, the membranes were incubated with either anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (Thermo Scientific, Rockford, IL, USA) for 1 h, and visualized using the FUSION-SOLO imaging system (Vilber Lourmat, ZAC de Lamirault, France). Antibodies against BAX, Bcl-2, p21, p53, PARP-1, caspase-3, caspase-7, caspase-8, caspase-9, HDAC1, HDAC2, HDAC3, and HDAC8 were purchased from Santa Cruz Biotechnology. Antibodies against acetylated-p53 (K373, K381, and K382) and ERα were obtained from Merck Millipore (Darmstadt, Germany). Anti-β-actin antibodies were bought from Sigma-Aldrich (St. Louis, MO, USA).