Figure 9

The C-terminal SREBP-1a region is essential for SFaN-mediated SREBP-1a degradation and ubiquitination. (A) Huh-7 cells were transfected with pCMV-SREBP-1a-(2–1147)-3 × FLAG, pCMV-SREBP-1a-(479–1147)-3 × FLAG, pCMV-SREBP-1a-(2–594)-3 × FLAG, pCMV-SREBP-1a-(2–968)-3 × FLAG, or pCMV-SREBP-1a-(2–784)-3 × FLAG and cultured in medium B for 24 h. The cells were trypsinized, seeded in six-well plates, and cultured in medium B for 24 h. The cells were then shifted into medium B in the presence of the vehicle, 100 μM SFaN or SFeN. After incubation for 3 h, whole-cell extracts underwent immunoblotting (IB) with anti-FLAG or anti-β-actin antibodies. The signals were quantified by Image J and normalized by β-actin signals, and the signals of the control group treated with DMSO were represented as one (n = 2–4). Different superscript letters denote statistical significance (p < 0.05). Full-length images of all replicates are presented in Supplementary Fig. S8. (B) Huh-7 cells were transfected with pCMV-SREBP-1a-(479–1147)-3 × FLAG or pCMV-SREBP-1a-(2–594)-3 × FLAG and HA-Ub and cultured in medium B for 24 h. The cells were trypsinized, seeded in 100 mm dishs, and cultured in medium B for 24 h. After incubation with 10 μM MG132 for 30 min, the cells were shifted into medium B supplemented with 10 μM MG132 in the presence of the vehicle, 100 μM SFaN, or SFeN. After incubation for 3 h, cell lysates were subjected to immunoprecipitation (IP) using an anti-FLAG antibody. The immunoprecipitates were subjected to immunoblotting (IB) with anti-HA or anti-FLAG antibodies. Full-length images are presented in Supplementary Fig. S9.