FIG. 6.

RA-induced PKC-CREB activation is mediated by the Raf/MEK/ERK/RSK signaling pathways. NHTBE cells were transfected with PKCα-siRNA (A), ERK-siRNA (B, left), RSK-siRNA (B, right), or a nonspecific control pool (NS-siRNA) as described in Materials and Methods. Three days after transfection, the cells were further treated with or without RA for 30 min. After the treatment, equal amounts of whole-cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies. All figures are representative of three independent experiments. β-Actin expression was used as an internal control. NT, nontransfection. (C) To determine the effects of siRNA on CRE transactivation, cells were cotransfected with a CRE-luciferase vector and siRNA of PKCα, ERK, RSK, or CREB. After transfection, cells were incubated with or without RA for 48 h. Cell lysates were then assayed for luciferase activity. The data represent luciferase units normalized according to β-gal in the same cell lysates. Values are means ± SE of triplicate experiments, and the figures are representative of three independent experiments.