Figure 4. Insulin and EGF induce the phosphorylation of FLNc at Ser²²¹³ in C2C12 cells.

(A) Murine C2C12 myoblasts were deprived of serum for 12 h, then stimulated with 250 ng/ml insulin or 20 ng/ml EGF for the times indicated. The cells were lysed and the lysates subjected to SDS/PAGE followed by transfer to an ImmobilonP membrane. The membranes were then probed with the anti-pSer²²¹³ and anti-FLNc antibodies, and with further antibodies that recognize PKBα phosphorylated at Ser⁴⁷³ (p473) and the phosphorylated and unphosphorylated forms of PKBα equally well (PKBα). (B) Cardiac muscle extracts (10 μg of protein) were prepared from either wild-type or PDK1 conditional knockout duplicate individual mice that had been fasted for 12 h before injection with or without insulin (10 m-units/g) for 20 min. Extracts were subjected to SDS/PAGE, transferred to ImmobilonP membranes and immunoblotted with antibodies as in (A), except that phosphorylation of PKBα was detected with an antibody that recognized PKBα phosphorylated at Thr³⁰⁸ (pT308).