Ccnd2

Frozen heart tissues were lysed in cold RIPA buffer with protease inhibitor (Sigma, 4693116001) and were homogenized with prechilled TissueLyser (Qiagen, 25/s, 2 mins, 3 cycles) by adding metal beads. After centrifuge, supernatants were collected to detect proteins with small molecular weight like Yy1, Ccnd1 and Ccnd2. 20 µg of each sample was loaded into SDS-page gel and further transfer to nitrocellulose membrane (0.2 µm, Biorad, 162-0112) for blotting. Primary antibody including: Yy1 (Thermofisher, PA5-29171), Ccnd1 (Abcam, ab16663), Ccnd2 (Cell Signaling Technology. Inc, 3741), and Lamin B1 (Abcam, ab16048). Secondary antibody including: Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (Thermofisher, A16035).

At 72 h after transfection, aliquots of 5 × 10⁶ cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology, China), and the lysate was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). A total of 30 μg protein per lane was resolved by 10% SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, USA). The membrane was blocked with 5% non-fat milk and then incubated overnight with primary antibodies against β-actin (Sigma-Aldrich, St. Louis, Missouri, USA), CDK-4 (Cell Signaling Technology, USA), CCND1 (Cell Signaling Technology, USA), or CCND2 (Cell Signaling Technology, USA). The membrane was washed in TBST and incubated with the secondary goat anti-mouse IgG antibody (Beyotime Institute of Biotechnology, Shanghai, China). An enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) was used to detect the protein bands using a FluorChem E System (ProteinSimple, CA, USA).

The cells were lysed using the mammalian protein extraction reagent RIPA (Beyotime, Beijing, China). Approximately a 50 μg protein extraction was separated by 10% SDS-PAGE, transferred to 0.22 mm nitrocellulose (NC) membrane (Sigma), and incubated with specific antibodies. Autoradiograms were quantified by densitometry using Quantity One software (Bio-Rad, CA, USA). β-actin (diluted 1:1000) antibody was used as a control and rabbit anti-FBXW7 (1:1000 dilution), p14 (1:100 dilution), p16 (1:100 dilution), p21 (1:150 dilution), p27 (1:50 dilution), CDK2 (1:200 dilution), CDK4 (1:100 dilution), CDK6 (1:100 dilution), c-myc (1:150 dilution), CCND1 (1:100 dilution), CCND2 (1:150 dilution), CCND3 (1:100 dilution), CCNE1 (1:150 dilution), CCNE2 (1:50 dilution) were provided by Cell Signaling Technology (MA, USA).

Proteins from the total cell lysates were separated by 10% SDS-PAGE and probed with different primary antibodies against MCL-1, CCND2, and GAPDH (Cell Signaling Technology, MA, USA).