Analyzing EZH2 Stability in Cell Lysates

Cell lysates were prepared in RIPA buffer (1 × PBS, 1% Nonidet P40/Triton X-100, 0.5% sodium deoxycholate, 2 mM EDTA, with or without 0.1% SDS).^{2 (link)} Antibodies used were: anti-H3K27me3 (1:10 000, 07-499, Millipore, Billerica, MA, USA), anti-H3 (1:10 000, Ab1791, Abcam, Cambridge, MA, USA), anti-Skp2 (1:500, sc-7164, Santa Cruz, Dallas, TX, USA), anti-EZH2 (1:200, 07-1583, Millipore), anti-EZH2 (1:1000, 5246, Cell Signaling, Danvers, MA, USA), anti-EZH2 (1:1000, 612666, BD Biosciences, San Jose, CA, USA), anti-β-actin (1: 10 000, A5316, Sigma, St Louis, MO, USA), ant-Flag M2 affinity gel (A2220, Sigma), anti-Flag M2 antibody (1:1000, F1804, Sigma), anti-C-Myc (1:1000, sc-40, Santa Cruz), anti-HA.11 (1:1000, MMS-101 P, Covance, San Diego, CA, USA), anti-TRAF6 (1:1000, sc-8409, Santa Cruz), and anti-TRAF6 (1:1000, 8028, Cell Signaling). To determine the stability of EZH2, cells were cultured in starvation medium with cycloheximide (CHX, 100 μg/ml). After that, cell lysates were harvested at indicated time points, and subject to western blotting analysis using EZH2 antibody.^{2 (link)}