Restoration of Sin1-/- MEFs with GFP-Sin1 expression vectors.

<p>A. Sin1 isoforms exert distinct functions in restoration of mTORC2 signaling. Sin1-/- MEF cells transfected with GFP empty vector, GFP-Sin1α, GFP-Sin1β, GFP-Sin1γ, GFP-Sin1δ respectively were grown in starved, or starved then restimulated with insulin or serum for 15min. Total cell lysates were analyzed for indicated proteins by immunoblotting. B. The MAPK pathway is not altered in Sin1 isoform-rescued Sin1-/- MEF cells. Sin1-/- MEF cells transfected with GFP empty vector, GFP-Sin1α, GFP-Sin1β, GFP-Sin1γ, GFP-Sin1δ respectively were grown in starved, or starved then restimulated with insulin or serum for 15min. Total cell lysates were analyzed for indicated proteins by immunoblotting. Single-letter abbreviations for the treatments are as follows: S, starvation for 12hr, I, insulin for 15min after starvation, F, FBS for 15min after starvation. C. The quantifications analysis of the phosphor-PKC band are shown.