ULK1 is a substrate of Caspase-3. (A) Protein levels of ULK1 and phosphorylated ULK1 (p-ULK1) in AE9a-expressing fetal liver cells from WT and Caspase-3 KO mice. (B) Protein levels of ULK1 in AE9a-WT cells treated with oridonin (0, 2.5, and 5 μM) for 24 hours. (C-D) In vitro cleavage assays of ULK1 by recombinant Caspase-3. 293T cell were transfected with pcDNA3 and pcDNA3-Flag-ULK1. Flag-ULK1 protein was immunoprecipitated by Flag M2 beads. The precipitated Flag-ULK1 was incubated with or without recombinant active Caspase-3 (0.2 μg) in the presence and absence of 4 mM DEVD (Caspase-3 inhibitor) at 25°C for 2 hours. The samples were analyzed by western blots using anti-Flag (C) or anti-ULK1 (D) and Caspase-3 antibodies. (E) Caspase-3 cleaves ULK1 at D485. In vitro cleavage assays of ULK1-WT and ULK1-mutates by Caspase-3 were performed. 293T cell were transfected with pcDNA3-Flag-ULK1-WT and pcDNA3-Flag-ULK1 mutants were overexpressed in 293T cells (top panel). Flag-ULK1 proteins were immunoprecipitated by Flag-M2 affinity gel. The precipitated Flag-ULK1-WT and Flag-ULK1 mutants were incubated with or without recombinant active Caspase-3 (0.2 μg) at 25°C for 2 hours. The cleaved samples were analyzed by western blots using anti-ULK1 antibodies and anti-Tubulin antibody (bottom panel).