Western blot was performed referring to our previous articles 24 (link)-26 (link). In brief, proteins from cultured cells or homogenized left ventricles were separated by 10% SDS-PAGE and were electrophoretically transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA; No. IPFL00010). Membranes were blocked with 5% nonfat milk for 60min at room temperature, and were subsequently probed with indicating primary antibodies overnight at 4°C, followed with the secondary antibodies at 37°C for 1 hour. The bands were scanned and quantified by Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), and protein expressions were normalized to total proteins or GAPDH. Nuclear and cytosolic protein fractions were separated by a commercial kit as our previously described 9 (link), 21 (link). Proteins from cytosolic lysates were normalized to GAPDH, whereas proteins from nuclear lysates were normalized to PCNA.
Total RNA was isolated using TRIzol according to our previous studies and reverse transcribed with Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA)27 (link). The expression level of each individual transcript was normalized to Gapdh.
Total RNA was isolated using TRIzol according to our previous studies and reverse transcribed with Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA)27 (link). The expression level of each individual transcript was normalized to Gapdh.
