Figure 4.

Autophagy induction by synthetic, natural or physiological inducers disrupts the BECN2-GPRASP1 interaction in vitro and in vivo, dependent on the upstream ULK1 kinase complex. (A) Co-immunoprecipitation (co-IP) of endogenous human GPRASP1 with FLAG-human BECN2 in HEK293 cells treated with normal or starvation medium, or normal medium with ML246 for 3 h. The same co-IPs were also performed in HEK293 cells transfected with siRNAs against Atg13, Rb1cc1 or Ulk1. The lower panel shows the siRNA knockdown efficiency of each gene. (B) Co-IP of endogenous GPRASP1 with endogenous BECN2 using brain lysates from WT mice treated with vehicle, ML246 or Rg2, or with 48-h starvation. Lysates in each group were pooled from 3 mice.