Figure 3. Role of AMPK and MTOR in the CASP2-mediated modulation of autophagy. (A) Western blotting was performed to detect the phosphorylation status of PRKAA, MTOR, RPS6KB, and EIF4EBP1 vs. total (unphosphorylated) protein in WT and casp2^−/− MEFs. The same blots were reprobed for GAPDH or tubulin, α (TUBA) that served as loading controls. (B) Densitometric analysis was performed using ImageJ to determine the expression levels of active (phosphorylated) vs. total (unphosphorylated) PRKAA, MTOR, RPS6KB and EIF4EBP1 and the values are expressed as active vs. total and the values were further normalized as percent of loading control. Error bar represents ± SEM. Statistical significance was determined by the Student t test. **, P ≤ 0.01, *, P ≤ 0.05, NS, P ≥ 0.05, the experiment was repeated 3 times. (C) casp2^−/− MEFs were transfected with siRNAs specific for Prkaa1 and Prkaa2. The representative western blot demonstrates the efficiency of siRNA-mediated downregulation of PRKAA1 and PRKAA2, as assessed by western blot analysis. Western blotting for LC3 demonstrates the effect of Prkaa1 and Prkaa2 siRNA on loss of CASP2-mediated autophagy. (D) Effect of 500 nM rapamycin (inhibitor of MTOR) on autophagy in WT and casp2^−/− MEFs. Autophagy was detected by LC3 (an increase in LC3-II levels). Shown are the representative blots; the experiment was repeated at least 3 times.