Fig. 4.

cAMP stimulates the recruitment of SF1 and SREBP1 to the DGKθ promoter. A: H295R cells were incubated with 0.4 mM Bt₂cAMP, cross-linked with formaldehyde, and the sheared chromatin immunoprecipitated with antibodies against anti-SF1, anti-acetyl histone H4, or anti-SREBP1 and recruitment to the DGKθ promoter (−1,000/−700) assessed by qPCR and normalized to the ΔCt values of input DNA. Data are expressed as fold change over untreated control and represent the mean ± SD of four separate experiments, each performed in duplicate. A representative gel of PCR reaction is shown where reactions were subjected to agarose (2%) gel electrophoresis and the EtBr-stained PCR products (top bands are output and lower bands input) imaged using a VersaDoc scanner (Bio-Rad). B: CV1 cells were transfected with expression plasmids for GFP-tagged SF1 and FLAG-tagged SREBP1a or SREBP1c and harvested 48 h after transfection. Lysates were subjected to IP using an anti-FLAG antibody and protein A/G agarose. Immobilized proteins were washed, separated by SDS-PAGE, and analyzed by Western blotting. Blots were hybridized to anti-SF1 (upper and lower panel) or anti-FLAG (5% of input lysates; middle panel) antibodies. Shown are representative blots of coimmunoprecipitation experiments that were performed on five separate occasions, each time in duplicate.