A32961

Tissue or cell extracts were prepared in RIPA lysis buffer (89900, Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitor (A32961, Thermo Fisher Scientific). Lysates were cleared by centrifugation. Supernatant proteins were mixed with LDS Sample Buffer (NP0007, Thermo Fisher Scientific), separated by SDS-PAGE and transferred to nitrocellulose membranes (1704270, BioRad, Hercules, CA, USA). Membranes were probed with the following primary antibodies: anti-SMYD2 (sc-393827, Santa Cruz Biotechnology, Dallas, TX, USA), anti-TMPRSS2 (ab109131, Abcam, Cambridge, UK), and HRP-linked β-actin (ab49900, Abcam).

C2C12 cells were seeded at 2 × 10⁵ cells/cm² into a 6 cm dish, and cell lysates were collected after VV stimulation from days 1 to 3. RIPA Lysis and Extraction Buffer (89900, Thermo Fisher Scientific Inc., Waltham, MA, USA) containing protease and phosphatase inhibitor (1:100; A32961, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to extract the cell lysate. Western blot analysis was performed with primary antibodies against stathmin (1:1000), decorin (1:1000), Col-I (1:500), FGF7 (1:1000), TGFBr1 (1:500) and PAK3 (1:500) (Abcam Inc., Cambridge, MA, USA), pAKT (1:500), AKT (1:2000), MyoD (1:1000) and myogenin (1:500) (Cell Signaling Inc., Danvers, MA, USA), β-actin (1:10,000) (Arigo Biolaboratories Corp., TE Huissen, Netherlands), which were incubated with the membranes at 4 °C overnight. After being washed 3 times for 15 min each, the membranes were incubated with an HRP-conjugated goat antibody anti-mouse or anti-rabbit IgG secondary antibody (Arigo Biolaboratories Corp., TE Huissen, Netherlands) for 1 h at RT. The proteins were detected using an ECL substrate kit (Abcam Inc., Cambridge, MA, USA). Band intensity was quantified by using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Intensity data were normalized to GAPDH and indicated controls for the target protein fold changes calculation.