Figure 2.
BECN1 is a physiological target of AMPK. (A) HEK293T cells were cotransfected with plasmids encoding GFP-PRKAA1WT and GST-BECN1. GST-BECN1 was immunoprecipitated using glutathione agarose beads and detected with immunoblots using the indicated antibodies. (B) GST-BECN1 was affinity isolated in various PRKAA1 mutant-expressing HEK293T cells. Phosphorylated BECN1 was detected using p-AMPK substrate antibody and anti-GST to test GST-BECN1 expression. Glucose starvation (3 h) (C) or AICAR treatment (0.25 mM, 1 h) (D) were performed in pCDH1-Flag- BECN1-transfected HEK293T cells. p-AMPK substrate antibody was used to detect the p-BECN1. Whole cell lysates were immunoblotted with anti-Flag, p-AMPK, PRKAA and ACTIN antibodies. (E) After transfection with Flag-BECN1, HEK293T cells were with treated with compound C (Com. C), followed by glucose deprivation for 3 h. Flag-BECN1 was immunoprecipitated and immunoblotted with p-AMPK substrate antibody. Expression of Flag proteins was used as a loading control. IP, immunoprecipitation; WCL, whole cell lysate.