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Fig. 4. Alanine scanning mutagenesis of human Insig-2 reveals five amino acid residues crucial for 25-HC mediated inhibition of SREBP-2 processing in insect cells. (A) Amino acid sequence and predicted topology of human Insig-2. The topology is based on that of Insig-1 (26). Amino acids in blue denote residues that, when mutated to alanine, did not interfere with 25-HC-mediated inhibition of SREBP-2 cleavage. Amino acids in red denote residues that when mutated to alanine created resistance to 25-HC mediated inhibition of SREBP-2 cleavage. (B) Immunoblot analysis of SREBP-2 cleavage. Drosophila S2 cells were transfected with the following plasmids per dish: 0.4 µg of pDS-HSV-SREBP-2, 0.05 µg of pAc-Scap, and 0.05 µg of either wild-type pAc–Insig-2–Myc or the indicated mutant. On day 2, the cells were switched to medium F containing the indicated concentration of 25-HC. After incubation for 6 h at 23°C, cells were harvested and whole cell lysates were subjected to SDS/PAGE and immunoblot analysis with anti-HSV-IgG (against SREBP-2), IgG-9D5 (against Scap), and IgG-9E10 (against Insig-2). P and I denote the uncleaved precursor and cleaved intermediate forms of SREBP-2, respectively.