Figure 7.

hVps34 dissociates from 14-3-3 and is activated during starvation-induced autophagy. (a) HeLa cells expressing GFP-LC3 left untreated or starved for indicated times were analyzed using GFP-LC3 by microscopy. Bars=10 μm. Quantification of number of GFP-LC3 punctae per cell was performed by counting in a blinded experiment. Columns, average of three different experiments, set up at duplicated samples and counting 6 × 100 cells per sample (N=3, ^*P=0.0047, Student t-test; left panel). Untreated and starved HeLa cells were harvested for immunoblot analysis of indicated proteins. Tubulin was used as a protein loading control (right panel). (b) HeLa cells were treated as in (a) and extracts from untreated and starved 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 test presence of hVps34 protein in immunoprecipitates and 14-3-3 binding (DIG-14-3-3) (top panel: quantification of hVps34 in vitro kinase assay, N=3, ^*P=0.0001, Student t-test). A full-color version of this figure is available at Cell Death and Differentiation online