Anti myogenin sc 12732

The cells grown under each culture condition were washed by cold phosphate‐buffered saline and were directly harvested with 1× SDS sample buffer. The cells were sonicated by Branson Sonifier 450 (Branson Ultrasonics, Danbury, CT, USA). The homogenate (5–20 μL of each sample) was separated on SDS/PAGE gels for western blotting with anti‐DGKδ [9 (link)], anti‐cyclin D1 (sc‐450, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐cyclin D3 (sc‐182, Santa Cruz Biotechnology), anti‐myogenin (sc‐12732, Santa Cruz Biotechnology), anti‐myosin heavy chain (MyHC; MF20, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti‐β‐tubulin (T4026, Sigma–Aldrich), anti‐β‐actin (A5441, Sigma–Aldrich) and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; 016‐25523, Wako Pure Chemicals) antibodies. The immunoreactive bands were visualized using horseradish peroxidase‐conjugated anti‐rabbit or anti‐mouse IgG antibody (Cell Signaling Technology, Danvers, MA, USA) and Pierce™ ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA). The intensity of each band was measured using Amersham™ ImageQuant™ 800 (GE Healthcare, Chicago, IL, USA).

Proteins from the cell lysates were prepared in 100 mg/mL RIPA lysis buffer containing 1% (v/v) phenylmethylsulfonyl fluoride (Beyotime, China), then centrifuged at 12,000 g for 10 min at 4 °C. After that, the lysates were heated at 95 °C for 5 min in 5× sodium dodecyl sulfate (SDS) sample buffer. Identical quantities of proteins were separated by 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), and non-specific binding was blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 for 4 h. The membranes were incubated overnight with primary antibodies at 4 °C, washed three times, and then incubated with the secondary antibodies for 1 h at 37 °C. The antibodies for Western blot analysis were obtained from Santa Cruz Biotechnology, including anti-myogenin (sc-12732; 1:200 dilution), anti-MyoD (sc-760; 1:200 dilution), anti-MyHC (sc-376157; 1:1000 dilution), and anti-β-actin (sc-4777; 1:1000 dilution). Changes in protein levels were normalised to the housekeeping protein β-actin for quantitative Western blot analysis using ImageJ software.