Figure 5. Identification of the VMP1-Beclin 1-hVps34 complex.

(a) Coimmunoprecipitation assays. Lysates from HeLa cells transfected with pSG5-Flag-Beclin 1 and pcDNA4-VMP1^ΔAtgD or pcDNA4-VMP1 expression plasmids incubated with anti-Flag covalently bound to magnetic beads. After washes, eluted proteins were separated and subjected to anti-Beclin 1, V5 and hVps34 immunoblotting. (b) Coimmunoprecipitation assays. Lysates from HeLa cells transfected with pCR3.1-Flag-Beclin 1-BD and pcDNA4-VMP1 expression plasmids incubated with anti-Flag covalently bound to magnetic beads. After washes, eluted proteins were separated and subjected to anti-Flag, V5 and hVps34 immunoblotting. (c) Coimmunoprecipitation assays. Lysates from HeLa cells treated with rapamycin were incubated with anti-Beclin 1 antibody. Eluted proteins were subjected to anti-Beclin 1, anti-VMP1 or anti-hVps34 antibody. (d) Coimmunoprecipitation assays. Lysates from HeLa cells treated with rapamycin were incubated with anti-VMP1 antibody. Eluted proteins were subjected to anti-VMP1, anti-Beclin 1 or anti-hVps34 antibody. (e) HeLa cells transfected with a plasmid harboring pcDNA4-VMP1-V5 were coprecipitated using nickel-agarose beads. pcDNA4-VMP1^ΔAtg expressing cells were used as control. The precipitates were subjected to SDS-PAGE and silver nitrate staining. Data is representative of three independent experiments.