Overexpression of Insig-1 increases sensitivity of SREBP processing to inhibition by dietary cholesterol. Eight-week old female mice (four per group) were fed an ad libitum chow diet supplemented with the indicated amount of cholesterol for 2 days prior to study. The WT mice were littermates of the transgenic mice. A detailed description of these mice is provided in Supplemental Table 1. (A) Immunoblot analysis. Livers from each group (four mice per group) were pooled, and 30-μg aliquots of the membrane and nuclear extract fractions were subjected to SDS-PAGE and immunoblotted with Ab’s against SREBP-1 and SREBP-2. Chol., cholesterol; N, nSREBP; P, precursor form of SREBP. Asterisks denote nonspecific bands. CREB (cAMP-responsive element binding) protein was used as a loading control for the nuclear extract fractions (9192; Ab from Cell Signaling Technology, Beverly, Massachusetts, USA). (B) The gels of nuclear extract fractions in A were scanned and quantified by densitometry. Intensities of the cleaved nuclear forms of SREBP-1 and SREBP-2 in lane 1 (WT mice fed with 0.02% cholesterol) were arbitrarily set at 100%. (C) Cholesterol content of the livers of WT and TgInsig-1 mice fed for 2 days with the indicated amount of cholesterol. Each value represents the mean ± SEM of data from four mice. The levels of statistical significance (Student’s t test) between the WT and TgInsig-1 groups are shown as P values.