Correlation of ERK1/ERK2 MAPK activation with PPF.

Cells were cultured in FN/PMA for 3 days and analyzed by Western blot. (A) Time course of ERK1/ERK2 phosphorylation in the cultures with the FN/PMA combination or with FN or PMA alone. The upper panels show that phosphorylation of ERK1/ERK2 was faintly expressed in the presence of FN alone after 1 day of culture. The middle panel shows that phosphorylation of ERK1/ERK2 was rapid and weakly expressed in the presence of PMA alone and diminished with time. The lower panel shows that phosphorylation of ERK1/ERK2 was strongly expressed only in the presence of FN plus PMA after 5 minutes' treatment and was sustained up to 3 days, correlating with PPF. (B) Effect of signal inhibitors on phosphorylation of ERK1/ERK2. The inhibitors were added 1 hour prior to addition of PMA for 5 minutes. The upper panel shows that phosphorylation of ERK1/ERK2 was inhibited by PD, an MEK inhibitor (10 μM and 50 μM*); GFX, a PKC inhibitor (5 μM and 25 μM*); and SB, a p38 and JNK MAPK inhibitor (50 μM*). No inhibition seen with SB (10 μM) and wortmannin (W), a PI3K inhibitor (100 nM and 500 nM*). SFM indicates serum-free medium. The data are representative of 3 separate experiments.