PI4KIIIβ interacts with ATG13. (A) HEK293A-GFP-ATG13 cells were treated with RF or PI4KIIIβ siRNA for 72 h and then incubated in ES for 2 h, fixed, and labeled using antibody to ULK1. Scale bars, 10 µm. (B) GFP-ATG13 puncta in cells treated as in A were counted. Statistical analysis using two-tailed unpaired Student’s t test, mean ± SEM, n = 3 experiments, 100 cells per condition per independent experiment were quantified; ***, P ≤ 0.001. (C) HEK293A stably expressing GFP-ATG13 and mRFP-ATG9A were transfected with iRFP-PI4KIIIβ, incubated in ES, and imaged live by Airyscan microscopy. Scale bars, 5 µm; inset, 1 µm. (D) GFP-TRAP pulldown of HEK293A cells transiently expressing GFP-PI4KIIIβ incubated in ES for 2 h followed by immunoblot for GFP and ATG13. (E) GFP-TRAP pulldown of HEK293A-GFP-ATG13 cells incubated in ES for 2 h followed by immunoblot for GFP, PI4KIIIβ, and pS294 PI4KIIIβ. In H and I, bottom panel is Ponceau S staining of input and GFP-TRAP.