Figure 2.

CHIP analysis for Sp1 association with LDL receptor promoter in sterol-depleted CHO-7 cells. (A) A schematic representation of the LDL receptor promoter showing the two required Sp1 sites and the single SREBP site upstream from the TFIID interacting region. All three of these sites are required for sterol regulation (28). (B) PCR analysis of CHIP reactions. Chromatin from cells cultured in the absence (I) or presence (S) of regulatory sterols was analyzed by PCR with LDL receptor promoter-specific primers, as described in Materials and Methods. The starting chromatin from each preparation was diluted 1:1,000, and 2 μl were analyzed in lanes 1 and 2. Samples were processed through the CHIP protocol, and the primary antibody was left out (lanes 3 and 4) or an Sp1 antibody was included (lanes 5 and 6). Twenty-eight cycles of PCR were used. (C) The input material from the induced (I) or suppressed (S) was serially diluted in 3-fold steps in lanes 1–3 to document that the amount of PCR product accurately reflects the amount of template DNA added to the PCR. (D) Equal aliquots of starting chromatin from each sample were subjected to SDS/PAGE and immunoblot analysis with the Sp1- or SREBP-2-specific primary antibodies, as indicated. P and M denote the positions of putative precursor and mature forms, respectively, of SREBP-2.