Figure 4.

hVps34 dissociates from 14-3-3 and is activated during C2-ceramide-induced autophagy. HeLa cells continuously growing in serum were left untreated or stimulated with C2-ceramide (10 μM) for 24 h to promote autophagy. (a) Extracts (2 mg) from untreated (U) or C2-ceramide (C2) stimulated HeLa cells were incubated with 1 μg of Beclin-1 (Santa Cruz Biotecnology, Inc.) antibody bound to protein-G-Sepharose. The washed immunoprecipitates were resolved using SDS-PAGE, transferred to nitrocellulose and probed with DIG-14-3-3 (14-3-3 overlay), Beclin-1 (BD Biosciences) and hVps34 (Zymed) antibodies. The asterisk (^*) shows an unknown protein band. The arrow corresponds to hVps34 size on DIG-14-3-3. The washed immunoprecipitates were also used for hVps34 in vitro kinase assay (right panel). Autoradiography shows levels of PI3P³². (Quantification of hVps34 in vitro kinase assay expressed as PI(3) fold change, N=3, ^*P=0.0007, Student t-test.) (b) Extracts (1 mg) from untreated (U) or C2-ceramide (C2) stimulated HeLa cells were incubated with 1 μg of hVps34 (Echelon Biosciences) antibody bound to protein-G-Sepharose. The washed immunoprecipitates were used for hVps34 in vitro kinase assay and also to analyze interaction with 14-3-3 (DIG-14-3-3) and presence of Beclin-1, hVps34 and 14-3-3 proteins in the immunoprecipitates (quantification of hVps34 in vitro kinase assay, N=3, ^*P=0.0007, Student t-test). Piece of the DIG-14-3-3 shown correspond to hVps34 size. (c) Extracts (30 μg) from HeLa cells continuously growing in serum or stimulated with C2-ceramide (10 μM) for 24 h to promote autophagy were run on SDS-PAGE, transferred to nitrocellulose membranes and probed with indicated antibodies