Ab6721

The total protein was extracted from AC16 cardiomyocytes using RIPA buffer (Beyotime, Shanghai, China) and the protein concentration was tested using the Bicinchoninic acid (BCA) protein quantification kit (Beyotime). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was employed to separate the proteins (30 μg/sample), and then the proteins in SDS-PAGE were transferred onto the polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin (BSA) for 1 h, membrane was cut according to the molecular weight of the target proteins and incubated with primary antibodies that were purchased from Abcam (Cambridge, MA, USA), including Cyclin D1 (1:200, ab16663), p21 (1:2000, ab109520), Bcl-2 (1:2000, ab182858), Bax (1:5000, ab32503), FUS (1:1000, ab124923), SIRT3 (1:1000, ab217319) and GAPDH (1:2500, ab9485) overnight at 4 °C. Subsequently, the membrane was incubated with the HRP-conjugated secondary antibody (1:10,000, ab6721) for 1 h. Enhanced chemiluminescence (ECL) reagent (Beyotime) were applied to visualize protein bands. The gray value analysis was evaluated by using Image J software.