Ab22553

Cells were lysed using SDS lysis buffer (containing 4% SDS and 100 mM Tris-HCl (pH=6.8)) and boiled at 105°C on a thermomixer for 10 min. Protein samples were diluted (ranging from 1:10 to 1:20) and protein concentration was measured by BCA kit. About 20 μg protein per sample was subjected to SDS-PAGE and electrotransferred to a PVDF membrane (Millipore). The membrane was blocked with 5% skim milk (powder from BBI Life Sciences) and incubated with primary antibodies for ∼12 hr at 4°C, then with horseradish peroxidase (HRP)-conjugated secondary antibodies. The visualization and data processing were performed by a ChemiDoc XRS system (Bio-Rad). Antibodies used in this study were as follows: anti-ZKSCAN3 (Santa Cruz, sc-515285), anti-HP1α (Cell Signaling Technology, #2616S) and anti-KAP1 (Abcam, Ab22553), anti-Lamin B1 (Abcam, Ab16048), anti-LBR (Abcam, Ab32535), anti-P16 (BD Bioscience, 550834), anti-P21 (Cell Signaling Technology, #2947), anti-β-actin (Santa Cruz, sc-69879), anti-Flag (Sigma, F1804) and anti-GAPDH (Sigma, G8795).

For Western analysis of histones, cell extracts were prepared by lysis in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.25% deoxycholate, 0.1% SDS). Extracts were boiled in SDS-PAGE loading buffer and blotting was performed as previously described using anti-TRIM28 (ab22553, 1:5000; Abcam), anti-H3S10ph (H. Kimura, CMA311, 1:5000), and anti-pan H3 (H0164, 1:5000; Sigma). Following addition of IRDYE-conjugated secondary antibodies, fluorescence was quantified on an Odyssey imager (LiCOR Biosciences).