Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Oxysterols block transport by binding to Insig

Mutant Insig-2s do not bind to Scap in presence of 25-HC or cholesterol. On day 0, Scap-deficient SRD-13A cells were set up in medium C at 3.5 × 10⁵ cells per 60-mm dish. On day 2, each dish of cells was transfected in medium B with 0.1 μg of pCMV-Scap and one of the following pCMV–Insig-2–Myc plasmids: wild-type, 0.16 μg; F115A mutant, 0.32 μg; or T136A mutant, 0.16 μg. After incubation for 6 h at 37°C, the cells were switched to medium D for 16 h, then switched to medium D containing 1% HPCD for 1 h, after which the cells were washed twice with PBS and switched to medium D containing no sterols, 0.075 μM 25-HC (in ethanol), or 30 μM cholesterol (complexed to MCD), as indicated. After incubation for 6 h at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal anti-Myc to precipitate Insig-2. Pellets and supernatants (2.5:1 ratio) were subjected to SDS/PAGE and immunoblot analysis. Sup, supernatant fraction; IP, immunoprecipitate; IB, immunoblot.