Sterol-dependent localization of SCAP. (A and B) Localization of stably expressed GFP-SCAP in HeLa cells with or without cholesterol depletion (Chol Depl) for 3 h or treated with 2 µg/ml 25-HC for 2.5 h. (B) Accumulation of GFP-SCAP in GM130 (cis-Golgi matrix protein)– and mannosidase II (cis/medial-Golgi marker enzyme)–positive membranes upon cholesterol depletion. N, nucleus. (C) Proteolytic activation of SREBP2 upon cholesterol depletion. Lysates of HeLa cells with or without cholesterol depletion for 3 h or treated with 2 µg/ml 25-HC for 3 h were analyzed by Western blotting with an anti-SREBP2 antibody. Arrowheads indicate the precursor (P) and mature (M) forms of SREBP2. Asterisk denotes nonspecific bands. (D) Localization of the stably expressed hamster SCAP WT and mutants in shSCAP HeLa cells with or without cholesterol depletion for 3 h. Merged images for the hamster SCAP WT or mutants (green) and the cis/medial-Golgi marker GPP130 (magenta) of the boxed areas are shown in the insets. Scale bars, 10 µm.