a: C2C12 myotubes were transfected with scramble control siRNA (siCTL) or SCP4 specific siRNA (siSCP4) for 48 h in DMEM containing 2% horse serum. The knockdown efficiency was confirmed by immunoblots.
b: At 48 h after transfection, C2C12 myotubes with siCTL or siSCP4 were switched to serum-free media (SF) for 36 h. The rates of proteolysis were calculated from the linear slopes between 24 and 36 h (mean±SEM, n = 3/group, *p < 0.05).
c: The mRNA level of Myostatin, Atrogin-1 and MuRF1 were assessed by RT-PCR in siCTL and siSCP4 myotubes, which were treated with 2% horse serum or serum-free media for 36 h (mean±SEM, n = 3/group, **p < 0.01).
d: The levels of p-Akt, p-FoxO1, p-FoxO3a were assessed by immunoblots in siCTL and siSCP4 myotubes that had been treated with 2% horse or serum-free media for 36 h. Note that serum depletion decreases p-Akt in both siCTL and siSCP4 cells, but this response did not affect the p-FoxOs in myotubes with SCP4 knockdown