FIGURE 9:

Serine substitution in the P-motif enhances transcriptional activation and secretion of ApoA-IV. (a) HepG2 cells were cotransfected in triplicate with 50 ng of pGL3-ApoAIVpr-LUC and 500 ng of plasmids expressing wt or P-motif mutants in the context of normal or S1P/S2P-ve backgrounds as indicated. After 48 h, cells were extracted and luciferase assays performed as described in Materials and Methods. Values represent mean fold activation over the control samples, with error bars indicating SDs. (b, c) HepG2 cells were transfected with plasmids expressing wt and P-motif mutants of CREB-H. Medium was replaced at 48 h with serum-free medium for a further 24 h. The medium was then collected from equivalent cell numbers, and secreted proteins were precipitated and analyzed by Western blot for ApoA-IV as described in Materials and Methods. (b) Cellular protein extracts harvested and analyzed for detection of the CREB-H variants (with rabbit anti–γ-tubulin as a loading control). (c) Corresponding levels of secreted ApoA-IV in the medium. FL indicates the CREB-H precursor form and Cl the cleavage product.