Starvation- and p53-induced degradation of TBC1D15.

<p>(A) Flag- TBC1D15 was expressed alone or with myc-p53 as indicated, followed by cell lysis and analysis by immunoblotting. (B) p53 transactivation function mediates destabilization of myc-TBC1D15. HEK-293A cells were transfected with myc-TBC1D15 and either empty vector or Flag-p53 (human), wild-type murine p53 or the transactivation-deficient point mutant p53D278N, followed by lysis and immunoblotting. (C) Cells were transfected with Flag-TBC1D15 and either empty vector or myc-p53, followed by exposure for 6 hr to autophagy inhibitor Bafilomycin A1 (100 nM) or proteasome inhibitor lactacystin (5 μM). (D) HEK-293A cells expressing myc-TBC1D15 were exposed for 6 hr to DMSO vehicle or to the mTOR inhibitors rapamycin (100 nM) or PP242 (1 μM), then lysed and analyzed by immunoblotting. (E) Cells were transfected with myc-TBC1D15 and cultured in complete media or shifted to glucose- and amino acid-free media for 6 hr in the absence or presence of Bafilomycin A1, as indicated. (F) Elevated levels of TBC1D15 in <i>Atg5</i>- and <i>p53</i>-deficient livers. Livers were surgically resected from p53-deficient mice or from mice with hepatocyte-specific deletion of <i>Atg5</i> (<i>Atg5^fl</i>^/fl; <i>Alb-cre</i>) or littermate controls. Liver lysates were resolved by SDS-PAGE and analyzed by immunoblotting.