a) The mTORC2 complex integrity in Sin1+/- (WT) or Sin1-/- (KO) Ab-MuLV pre-B cells was determined by immunoprecipitation (IP) of Rictor followed by immunoblotting (IB) of Sin1, Rictor and mTOR.
b) Akt phosphorylation and expression was measured in primary Sin1+/+ or Sin1-/- pro-B cells cultured on OP9 cells with IL-7 and Ab-MuLV transformed Sin1+/- or Sin1-/- pre-B cells. Samples were normalized to total cellular protein and blotted as indicated.
c) FoxO1 phosphorylation and expression was measured in Sin1+/- or Sin1-/- Ab-MuLV pre-B cells. Samples were normalized to total cellular protein and blotted for FoxO1 or FoxO1 p-Thr24.
d) FoxO1 phosphorylation was measured in Sin1+/+ or Sin1-/- pro-B cells cultured with serum and IL-7. The samples were normalized to total FoxO1 protein then blotted for FoxO1 p-Thr24.
e) FoxO1 expression was measured in Sin1+/+ or Sin1-/- pro-B cells. The samples were normalized to total cellular protein.