FIG. 5.

SREBP-2 binds to the S3 and S5 sites of the caspase-2 promoter. Chromatin was prepared from untreated HepG2 cells (−) or cells treated with 50 μM lovastatin (+) for 36 h. Cells were incubated with ALLN for the last 3 h before harvesting to stabilize the nuclear SREBP-2 form. Each cross-linked chromatin extract was incubated with anti-SREBP-2 antibody (S2), with negative control IgG (IgG), or without antibody (−). DNA extracted from each immunoprecipitate was analyzed by PCR using the relevant primers (see Materials and Methods; also see Table S4 in the supplemental material) to test the binding of SREBP-2 to the S2, S3, and S5 sites from the CASP-2 gene and on the SRE-binding sequences of the FPP synthase gene as a positive control. The input chromatin (I) corresponds to a 1:100 dilution of total DNA extracted prior to immunoprecipitation. One representative experiment of two independent experiments with similar results is shown. Optimization of PCR amplification parameters (PCR cycle numbers and input DNA amount) was performed in pilot experiments.