Figure 2.

BECN1s is not involved in macroautophagy initiation. (A) HEK 293T cells were transfected with control vector, Flag-BECN1, or Flag-BECN1s as indicated. Twenty-four h later, cell lysates were immunoprecipitated with anti-Flag antibody, followed by western blot analysis with anti-PIK3C3 antibody. (B) HeLa cells with BECN1 stable knockdown were transfected with GFP-2*FYVE plus either control vector, Flag-BECN1, or Flag-BECN1s as indicated. Twenty-four h after transfection, cells were immunostained with anti-Flag antibody. The images were taken under a fluorescence microscope. Scale bar: 20 μm. (C) HeLa cells expressing control shRNA, BECN1 shRNA, or BECN1s shRNA were treated with EBSS for the indicated periods of time. Cell lysates were then analyzed by western blot with anti-SQSTM1 and anti-GAPDH antibodies. The shRNA-mediated knockdown efficiency for BECN1 and BECN1s was also shown. (D) HeLa cells expressing the indicated shRNAs and proteins were treated with EBSS for the indicated periods of time. Cell lysates were then analyzed by western blot with anti-SQSTM1, anti-Flag and anti-GAPDH antibodies. (E) HEK 293T cells were transfected with either control vector, Flag-BECN1 or Flag-BECN1s as indicated. Twenty-four h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody, followed by western blot analysis with anti-UVRAG and anti-Flag antibodies.