FIGURE 9:

CREB‑H phosphodegron mutants display enhanced functional activity on the apolipoprotein A IV target promoter. (a) Cells were cotransfected in triplicate with the apolipoprotein A IV–Luc target vector without or with 10 ng of the expression vectors for each of the CREB-HΔTMC protein as indicated. Samples were subsequently processed for luciferase activity. Results are plotted as absolute activity compared with reporter alone control, which was transfected with equal amount of pcDNA3 DNA. (b) Parallel samples from cells transfected with 100 ng of the corresponding vectors were analyzed for expression levels of each of the CREB-H variants. (c) Each of the mutants was incorporated into full-length proteins in isogenic expression vectors, which were transfected into HepG2 cells. The cells were solubilized and the cleaved nuclear product analyzed by Western blotting using fluorochrome-coupled secondary antibodies. Anti-actin was probed in parallel as a loading control. The N and N¹ species detected were entirely consistent with the levels observed from the independent cleaved product. (d) Cells transfected with the wild type or mutants as indicated were harvested and RNA prepared and analyzed for control marker RNAs (TBP and actin), CREB-H RNA, and apolipoprotein A IV RNA. Absolute levels of apolipoprotein A IV RNA were normalized for the endogenous marker TBP and for CREB-H transcript levels. (e) In further analyses, the medium from similarly transfected cells was harvested, precipitated, and analyzed for total levels of secreted apolipoprotein A IV as described in Materials and Methods.