Fig. 7.
Effects of oleic acid on phosphorylation of c-Raf in cultured HASM cells. Cells were stimulated with oleic acid, and subsequently cell lysates were processed to detect phosphorylated and total levels of c-Raf by immunoblot. For A–D: i: immunoblot analysis; ii: graphical analysis. A: effects of oleic acid (10 µM) on the phosphorylation of c-Raf at either Ser 338 (n = 6) or Ser 259 (n = 5) in HASM cells. B–D: effects of inhibitors of either Gαi or Gαq protein or Gβγ subunits on oleic acid-induced phosphorylation of c-Raf at Ser 338 in HASM cells. Cells were pretreated with the Gαi-specific inhibitor pertussis toxin (PTX; 100 ng/ml for 4 h; n = 5; B), Gαq-specific inhibitor YM-254890 (1 µM for 30 min; n = 8; C), or Gβγ signaling inhibitor gallein (10 µM for 30 min; n = 7; D) before treatment with oleic acid (10 µM) for 10 min. Phosphorylation of c-Raf is presented as a ratio of phosphorylated to total c-Raf and then normalized to basal levels. Data represent means ± SE. *P < 0.05; ***P < 0.001, compared with basal.