Ab207604

The radio immunoprecipitation assay (RIPA) (Takara, Dalian, China) was introduced to isolate the protein. The BCA protein detection kit (Junxin, Suzhou, China) was applied to measure the concentration of protein. The proteins (30 μg in each lane) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked in 5% fat-free milk and exposed to different primary antibodies at 4 °C overnight. The membranes were incubated with horseradish peroxidase-conjugated IgG. The immunoreactive protein bands were detected with a chemiluminescence (ECL) reagent (Junxin, Suzhou, China) and the Bio-Rad ChemiDocTM XRS system. β-Actin was measured as a loading control. The antibodies used in this study are listed below: The anti-PTEN antibody (Ab267787, Abcam, Cambridge, British), anti-Bcl-2 antibody (Ab32124, Abcam, Cambridge, British), anti-Bax antibody (Ab182734, Abcam, Cambridge, British), anti-β-Actin (Ab207604, Abcam, Cambridge, British) and Goat Anti-Rabbit IgG H&L (HRP) (Ab 6721, Ab207604, Abcam, Cambridge, British) were used according to the protocol.

Protein expressions were determined using Western blot assay [33 (link)]. Total protein was extracted using RIPA cell lysate buffer containing a protease inhibitor cocktail. The protein concentration was then measured using the BCA method. The proteins were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were then blocked with 5% BSA at room temperature for 50 min to eliminate nonspecific binding of primary and secondary antibodies. Primary antibody (cleaved-Caspase3 antibody, ab32042, 1:1000, Abcam; GAPDH antibody, ab9485, 1:1000, Abcam; PCNA antibody, ab18197, 1:1000, Abcam; CCND2 antibody, ab207604, 1:1000, Abcam;) was then added to the PVDF membrane and incubated overnight at 4°C. The next day, the membranes were probed with the corresponding HRP-labeled secondary antibody (ab7090, 1:1000, Abcam) by incubation at room temperature for 2 h. ECL chromogenic solution A and B were mixed, added dropwise to the position of the target band on the membrane, and photographed using ImageJ software.