Figure 4. CaMKIV regulates ubiquitin-mediated degradation of mTOR.
A, Primary peritoneal macrophages from C57Bl/6 or CaMKIV−/− mice were isolated, and total mTOR mRNA assayed by real time PCR (n=4 independent experiments). B, Primary peritoneal macrophages from C57Bl/6 (n=1) or CaMKIV−/− (n=2) mice were isolated and exposed to MG132 (10μM) for 4 hours. Total cellular lysate (10μg per lane) was then isolated and analyzed for the expression of mTOR and actin by immunblot (representative blot of n=3 independent experiments; N=3 WT and N=6 CaMKIV−/− mice total were used for all 3 experiments combined): lanes 1 and 2 (WT 1), lanes 3 and 4 (CaMKIV−/− 1), lanes 5 and 6 (CaMKIV−/− 2). C, RAW 264.7 cells were exposed to STO609 (10μM) and MG132 (10μM) for 4 hours. Total cellular lysate (10μg per lane) was isolated, and mTOR and actin expression were assessed by immunoblot (representative blot of n=3 independent experiments). D, RAW 264.7 macrophages were exposed to STO609 (10μM) and MG132 (10μM) for 4 hours, at which point total cell lysate was harvested, immunoprecipitated for ubiquitin, subjected to immunoblot analysis, and probed for mTOR (representative blot of n=3 independent experiments).