The AH domain of ARFIP2 is required for autophagy. (A) Schematic representation of human FL ARFIP2 showing the AH and BAR domain (BAR). An asterisk indicates the point mutation of phenylalanine to alanine in position 99 (W99A). (B) HEK293A CTRL or CrARFIP2 KO (clone 1) cells transfected with HA-ARFIP2 FL or HA-ARFIP2 W99A mutant were incubated in FM or ES without or with Bafilomycin A1 (EB) for 2 h before immunoblotting for HA, Actin, and LC3. (C) Quantification of B; statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test, mean ± SEM, n = 8 experiments; *, P ≤ 0.05; ***, P ≤ 0.001. (D) CTRL or CrARFIP2 KO (clone 1) cells transfected with HA-ARFIP2 FL or HA-ARFIP2 W99A mutant were incubated in ES for 2 h, fixed, and labeled using antibodies to HA and LC3B. Scale bars, 10 µm. (E) LC3B puncta in CrARFIP2 KO (clone 1) cells expressing HA-ARFIP2 FL or ARFIP2 W99A were counted. Mean ± SEM, n = 3 experiments, 100 cells per independent experiment; ***, P ≤ 0.001.