FIG. 7.

Inhibition of PI3-kinase/Akt signaling blocks PDGF-induced CREB loss in SMCs. Rat PA SMCs were plated on eight-well slides at 20% confluence. The cells were transferred from complete medium to DMEM containing 0.2% FCS for 24 h and then treated with the signaling inhibitors indicated to the left of each row for 30 min at the concentrations indicated in the legend to Fig. 5. PDGF was then added to a concentration of 25 ng/ml. Duplicate wells were left untreated. Medium, PDGF, and inhibitors were replaced every 24 h for 72 h. (A) Cells were fixed and subjected to immunostaining for total CREB using an AlexaFluor 594-conjugated secondary antibody. The cells were also mounted with VectaShield with DAPI to visualize nuclei. Figure shows representative fluorescent deconvolution photomicrographs at a magnification of ×400. Rapa., rapamycin; Cntrl, control. (B) Cytosolic (“C”) and nuclear (“N”) fractions were resolved on 10% polyacrylamide-SDS gels and transferred to PVDF membranes. The figure shows a representative Western blot for total CREB and a Coomassie blue-stained gel as a loading control. (C) Whole-cell lysates (25 μg protein) from cells treated with and without PDGF and/or Akt inhibitor were resolved on polyacrylamide-SDS gels and transferred to PVDF membranes. The figure shows a representative long-exposure Western blot for total CREB and a Coomassie blue-stained gel as a loading control. Positions of CREB and ubiquitinated CREB (Ub-CREB) are indicated in the figure.