The SCAP–SREBP complex functions in CARTS biogenesis. (A) Effects of SCAP knockdown and expression of the hamster SCAP WT or mutants on the expression levels of SREBP1 and SREBP2. The precursor (P), but not the mature (M), form of SREBP2 was detected in parental HeLa cells and shSCAP HeLa cells stably expressing the hamster SCAP WT or mutants. Expression levels of LDLR and HMGR were significantly increased by expression of the L315F mutant. (B) Interactions of FLAG-SREBP1a and FLAG-SREBP2 with Myc-OSBP, Sac1, and VAP-A, but not with RTN-4B or Bap31, in HEK 293T cells. (C) Interactions of FLAG-SREBP1a and FLAG-SREBP2 with GFP-SCAP, Myc-OSBP, Sac1, and VAP-A, but not with RTN-4B or Bap31. (D) siRNA-mediated knockdown of SREBP1 and/or SREBP2 in HeLa cells. Asterisks in B–D denote nonspecific bands. (E) Biogenesis of mKate2-FM4-PAUF–containing CARTS in control (Cont), SREBP1, and SREBP2 knockdown cells. 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 = 11 cells per condition; * P, < 0.05; ****, P < 0.0001; one-way ANOVA multiple comparison test). Scale bar, 10 µm. IB, immunoblotted; IP immunoprecipitate.