Acetyl histone h3 lys9 c5b11

Total protein was extracted from cell pellets by lysing in RIPA buffer (Sigma-Aldrich, Saint Louis, MO, USA) containing 1x protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). EZQ Protein Quantitation Kit (Molecular Probes, Eugene, OR, USA) was used to measure protein concentration. Forty micrograms of total protein were denatured and electrophoresed on Any kD^TM Mini-Protean TGX precast polyacrylamide gels (Bio-Rad, Hercules, CA, USA), electroblotted on nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), and probed with the respective antibodies against acetyl-histone H3 (Lys9; C5B11; 1:1000)) or β-actin (D6A8; 1:1000) purchased from Cell Signaling Technology (Danvers, MA, USA). The bound antibodies were visualized with horseradish peroxidase-linked anti-rabbit IgG (1:3000; Cell Signaling Technology, Danvers, MA, USA) and enhanced chemiluminescence (Amersham, Pittsburgh, PA, USA). β-actin in the corresponding cell lysates was used as an additional loading control.

Cells were harvested by lysis in radioimmunoprecipitation assay buffer and protease inhibitors sheared with a 26-gauge needle. Protein (30Ag) was separated by 10% PAGE, transferred to 0.45 Am Immobilon-P Transfer membranes (Millipore), and analyzed by Western blotting with anti-LC3, p62, UVRAG, IGFBP2 antibodies, Acetyl-Histone H3 (Lys9) (C5B11) Rabbit (untreated or TSA-treated (400 nM for 18 hours) using Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mAb) (Cell Signaling Technology). Loading levels were normalized using 1:2,000 antivinculin antibodies (Sigma) and densitometric analysis. To determine the amount of secreted IGFBP-2, conditioned medium obtained from cell lines cultured for 48 h in serum-free DMEM was used to perform Western blot analysis for IGFBP-2 as described above.