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. Author manuscript; available in PMC: 2013 Dec 28.
Published in final edited form as: Mol Cell. 2012 Nov 8;48(6):875–887. doi: 10.1016/j.molcel.2012.09.029

Figure 2.

Figure 2

IRS-1 turnover is defective in mTORC2-disrupted cells. A. WT and SIN1−/− were treated with 10 μM cycloheximide (CHX) for the indicated times. Decreasing amounts of pre-existing IRS-1 protein levels were analyzed by immunoblotting, quantitated and plotted relative to non-CHX treated (right panel). Results are expressed as mean ± S.E.M. B. WT and SIN1−/− MEFs were starved of serum in Met/Cys-free media then pulse-labeled with 35S-Met/Cys and chased for the indicated times. Cell lysates were immunoprecipitated with anti-IRS-1 and the labeled newly synthesized IRS-1 were analyzed by SDS-PAGE and visualized by autoradiography. Right panel, quantification of immunoblots; results are expressed as mean levels of labeled IRS-1 ± S.E.M. relative to 0 hr of chase. ** p<0.01 C. WT and SIN1−/− MEFs were treated with or without 20 μM MG132 for 4 hours prior to harvest. Total cell lysates (right panel) or immunoprecipitated IRS-1 (left) from lysates were analyzed by immunoblotting. See also Figure S1.