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. 2006 Sep 18;26(22):8281–8292. doi: 10.1128/MCB.00941-06

FIG. 5.

FIG. 5.

PGE2-induced FGF-9 expression is mediated by ERK1/2. (A) A representative picture shows the phosphorylation of ERK1/2 after EP3 agonist stimulation. Serum-starved cells were treated with 10 μM sulprostone (Sul) in the presence or absence of different concentrations of rottlerin, Gö6976, or GF109203 (GF) for 15 min (n = 5). Equal amounts of lysates were analyzed by SDS-PAGE and immunoblotted with antibodies against p-ERK1/2 (upper panel) and total ERK1/2 (lower panel). (B) Serum-starved cells were treated with different doses of PMA (10 to 100 nM) for 15 or 60 min. Equal amounts of lysates were analyzed by SDS-PAGE and immunoblotted with antibodies against p-ERK1/2 and total ERK1/2. (C) Serum-starved cells were transiently transfected with 4 μg or 8 μg of control vector (vector), the catalytic subunit of PKCδ (CD_PKCδ), or the regulatory subunit of PKCδ (RD_PKCδ), and levels of phosphorylated ERK1/2 and total ERK1/2 were determined as described above. (D) Serum-starved cells were treated as described in the legend to Fig. 4A. Equal amounts of lysates were analyzed by SDS-PAGE and immunoblotted with antibodies against p-ERK1/2 and total ERK1/2. (E) Stromal cells were transiently transfected with 2 μg of dominant-negative ERK1 or ERK2 (DNERK1 or DNERK2, respectively) or control vector and incubated for 48 h. Serum-starved cells were then treated with 1 μM PGE2 or 10 μM sulprostone (Sul) for another 12 h. Concentrations of FGF-9 transcripts were quantified by standard-curve QC-RT-PCR (n = 5). Asterisks indicate significant differences from data for the PGE2- and sulprostone-treated groups (P < 0.05). (F) Stromal cells were transiently transfected with control vector (Vector), dominant-negative ERK1 or ERK2 (DNERK1 or DNERK2, respectively), or dominant-negative ERK1 and ERK2 in combination (DNERK1+DNERK2) as described above. Equal amounts of lysates were analyzed by SDS-PAGE and immunoblotted with antibodies against FGF-9 (upper panel) and β-actin (lower panel). All the experiments were repeated at least four times, and the results were similar.