HEK 293 cells were stimulated (+) / not stimulated (-) with various AMPK activators. VPS34 was immunoprecipitated from 1mg HEK 293 cellular extracts using 1 ug of anti- VPS34[S672B]. ImmunoprecipitatesweresubjectedtoSDS-PAGEanalysisbeforebeing transferred to nitrocellulose membrane and immunoblotted with anti-VPS34 [S672B] or anti-VPS34 phospho Ser 282 [S375C]. Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG antibody (1 in 2500 dilution) and enhanced chemiluminescence.

Recombinant VPS34 wildtype or S282A was phosphorylated by AMPK in vitro and SDS-PAGE analysis before being transferred to nitrocellulose membrane and immunoblotted with various bleeds of anti-VPS34 phospho Ser 282 [S375C]. Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG antibody (1 in 2500 dilution) and enhanced chemiluminescence.