Anti cdk6

Western blot analysis was performed according to standard methods as previously described.27, 28 Total protein was extracted from cell pellets or fresh tissue after harvest. The blots were immunostained with primary and secondary antibodies. Anti‐TEF (1:500) was purchased from Abcam (Cambridge, MA); anti‐Ki67 (1:1000) was purchased from Cell Signalling (Danvers, MA); and other antibodies, including anti‐p‐AKT^Ser473 (1:500), anti‐p‐AKT^Thr308 (1:500), anti‐AKT (1:500), anti‐CDK4 (1:500), anti‐CDK6 (1:500,), anti‐cyclin D1 (1:500), anti‐p21^Cip1 (1:200), anti‐p27^Kip1 (1:200), anti‐p‐FOXO1^Ser256 (1:500), anti‐FOXO1 (1:1000), anti‐p‐FOXO4^Ser197 (1:500) and anti‐FOXO4 (1:500), were purchased from Sigma (St. Louis, MO). The membranes were stripped and reblotted with an anti‐α‐tubulin monoclonal antibody (1:1000; Abcam), which served as a loading control.

Western blotting was performed as previously described 20 (link). Briefly, protein extracts were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were incubated at 4 °C overnight with the following primary antibodies: anti-GAPDH (1:2000, Cell Signaling Technology); anti-p16 (1:1000, Abcam); anti-p21 (1:1000, Cell Signaling Technology); anti-CDK4 (1:1000, Cell Signaling Technology); anti-CDK6 (1:1000, Sigma); anti-CyclinD1 (1:1000, Cell Signaling Technology); anti-EZH2 (1:1000, Cell Signaling Technology); anti-LSD1 (1:1000, Cell Signaling Technology); anti-E2F1 (1:1000, Cell Signaling Technology); anti-H3K27 (1:1000, Cell Signaling Technology); anti-H3K4 (1:1000, Cell Signaling Technology); anti-Rb (1:1000, Cell Signaling Technology); anti-p-Rb ser-780 (1:1000, Cell Signaling Technology); and anti-p-Rb ser-795 (1:1000, Cell Signaling Technology). The membranes were then incubated with an anti-rabbit/mouse IgG secondary antibody (CST, USA). The blots were developed with ECL western blotting substrate (Pierce, USA).The bands on the membranes were detected using a Syngene G-Box and GeneSnap software (Syngene, Cambridge, UK).