I have evaluated protein synthesis rate in C2C12 myotubes using SUnSET method. 100 nM insulin or 10 mM Leucine were used as a positive control, but puromycin incorporation did not change in these treatments. The detail protocol is shown in below, so can you give me some advice?

C2C12 myoblasts were cultured in 2% HS DMEM (High glucose) for 5 days to induce differentiation. Differentiated myotubes were cultured in DMEM (High glucose) without HS for 16 hr (serum starvation). Then medium was changed to fresh DMEM (High glucose) containing 100 nM insulin or 10 mM leucine. After 1 hr treatment, 1 µM puromycin was added to the medium. Thirty min later, the cells were harvested using RIPA buffer.

What am I missing?