Rabbit anti human cdk4

Cells were treated with 100 μM SA12 for 48 hours and then lysed with RIPA lysis buffer (Sangon). Untreated cells served as control. The obtained proteins were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore Corp, Billerica, MA, USA). After blocking with 5% nonfat milk in Tris-buffered saline solution containing 0.05% Tween-20, the membranes were incubated with primary antibodies at 4°C overnight. And then, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase at room temperature for 2 hours. Signals were visualized by electrochemiluminescence detection, and the density of the blots was analyzed and quantified by Gel-Pro Analyzer 4.0 software. The primary antibodies used for Western blot analysis were rabbit antihuman MECP2, rabbit anti-human cyclin D1, rabbit antihuman CDK4, rabbit antihuman p16 (Abcam, Cambridge, MA, USA), and mouse antihuman β-actin (Cell Signaling, Danvers, MA, USA). The secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) and goat anti-mouse IgG (Zhongshanjinqiao, Beijing, China).