Figure 6.

Downregulation of endogenous 14-3-3ζ increase hVps34 activity. HeLa cells were transfected either with siRNA oligonucleotide targeting 14-3-3ζ or with a scrambled RNA oligonucleotide as described in Material ad Methods. After 48 h, extract of untransfected (C) or transfected either with siRNA 14-3-3ζ (14-3-3ζ) or scrambled siRNA (SC) 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 to analyze the presence of hVps34 protein in immonoprecipitates (top panel: quantification of hVps34 in vitro kinase assay, N=3, ^*P=0.038, ^**P=0.017, Student t-test). Extracts from untransfected cells (C) or transfected either with siRNA 14-3-3ζ (14-3-3ζ) or scrambled siRNA (SC) were harvested for immunoblot analysis of 14-3-3ζ isoform. Tubulin was used as a protein loading control (bottom panel)