ARFIP2 controls the cytoplasmic PI4P pool and PI4KIIIβ localization. (A) HEK293A CTRL or CrARFIP2 KO (clone 1) cells incubated in FM or ES for 2 h before immunostaining for PI4KIIIβ and GM130. Scale bars, 10 µm. (B) Quantification of PI4KIIIβ and GM130 colocalization in A. Mean ± SEM, n = 3 experiments, Pearson’s coefficient was measured in 30 cells per condition per independent experiment, statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test; *, P ≤ 0.05; ***, P ≤ 0.001. (C) HEK293A cells were incubated in FM or ES for 2 h, fixed, and labeled using antibodies to PI4P, ATG9A, and ARFIP2. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. Arrowheads indicate colocalized structures. (D) Line scans of C in FM (1) and ES (2). (E) CTRL or CrARFIP2 KO (clone 1) cells were incubated in FM or ES for 2 h before immunostaining for PI4P and GM130. Scale bars, 10 µm. (F) PI4P puncta in E 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.