SCAP regulates CARTS biogenesis in a cholesterol-dependent manner. (A) Schematic representation of the SCAP topology. SSD is highlighted in light blue. N, N-terminus; C, C-terminus. (B) Establishment of shSCAP HeLa cells stably expressing the hamster SCAP WT and mutants. IB, immunoblotted. (C) Recovery of PI4P turnover on expression of hamster SCAP WT or D451A/L452A, but not of Y234A, Y298C, or L315F. The graph shows determination of the Golgi PI4P levels in the indicated cells. Data are means ± SEM (n = 69–96 cells per condition; ****, P < 0.0001; one-way ANOVA multiple comparison test). (D) Recovery of the biogenesis of mKate2-FM4-PAUF–containing CARTS on expression of hamster SCAP WT or D451A/L452A, but not of Y234A, Y298C, or L315F. The graph shows the number of mKate2-FM4-PAUF–containing CARTS in the indicated cells at 15 min after the temperature shift to 37°C. Data are means ± SEM (n = 20 cells per condition; ****, P < 0.0001; one-way ANOVA multiple comparison test). Scale bars, 10 µm.