Anti pkap1

Cells were lysed in modified RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.5% NP-40, 0.1% SDS, 50 mM NaF, 2 mM sodium orthovanadate) supplemented with a protease inhibitor mix (Thermo Scientific, Rockford, IL, USA). Unless otherwise described, 30 μg of protein were resolved by SDS–polyacrylamide gel electrophoresis (PAGE), transferred, and immunoblotted with various antibodies. The antibodies used were anti-p21 (sc-397) and anti-p53 (sc-126) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-CHIP (C3B6) and anti-γH2AX (2577) from Cell Signaling Technology (Danvers, MA, USA); anti-ubiquitin (ab7780) and anti-ub-48-linked (ab140601) from Abcam (Cambridge, United Kingdom); mouse monoclonal anti-tubulin (Sigma-Aldrich); and anti-KAP1 and anti-p-KAP1 from Bethyl laboratories (Montgomery, TX, USA).

Cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5% Tween-20, 2% Igepal CA-630, 2 mM PMSF, 50 mM β-glycerophosphate (Merck) and protease inhibitor cocktail tablet (cOmplete Mini, Roche Diagnostics). Equal amounts of lysates were loaded into precast mini-gels (Invitrogen) and resolved by SDS–PAGE. Transfer of proteins onto nitrocellulose membranes and incubation with primary/secondary antibodies were performed according to standard procedures. Visualization of protein bands was achieved by fluorescence imaging on the Odyssey Clx system (LI-COR Biosciences). Antibodies and dilutions used were as follows: anti-WRN (1:5,000, Novus), anti-pKAP1 (Bethyl Laboratories, 1:100), anti-tubulin (1:5,000, sigma), anti-MLH1 (Cell Signaling Technology, 3515, 1:1,000), anti-MSH2 (Abcam, ab52266, 1:1,000), anti-GAPDH (Cell Signaling Technology, 5174, 1:5,000), IRDye 680RD Goat anti-Mouse IgG (1:2,000, LI-COR Biosciences), IRDye 800CW Goat anti-Rabbit IgG (1:2,000, LI-COR Biosciences), Goat anti-Rabbit Alexa Fluor 647 (ThermoFisher, A27040, 1:5,000).