PIGF-induced ERK phosphorylation in both THP-1 cells and monocytes. (A) PlGF-induced ERK-1/2 activity in THP-1 monocytes. THP-1 cells were incubated with RPMI containing 2% serum overnight. The cells were then incubated with fresh serum-free media for 2 hours and then treated with PlGF (250 ng/mL) for the indicated time periods. Where indicated THP-1 cells were preincubated with U73122 (1 μM), wortmannin (200 nM), PD98059 (10 μM), and Ab–Flt-1 (5 μg/mL) for 30 minutes before treatment with PlGF. Cell lysates were analyzed for ERK-1 and -2 activities by Western blot using antiphospho MAPK (Thr202/Tyr204) antibodies (upper panel, pERK-1/2). Blots were stripped and reprobed with antibodies specific for unphosphorylated ERK-1/2 (lower panel, ERK-1/2). The experiment shown is representative of 3 experiments. (B) PlGF-induced ERK-1/2 activity in peripheral blood monocytes. Peripheral blood monocytes (1 × 106/mL) were incubated in RPMI medium containing 1% FBS for 1 hour followed by treatment with PlGF (250 ng/mL) for 3 minutes. Prior to treatment, the cells were either incubated with wortmannin (200 nM) or PD98059 (10 μM) for 30 minutes. Cell lysates were prepared, and 10 μg of the protein was subjected to 10% SDS-PAGE. The blot was probed with phospho ERK-1/2 antibody (upper panel), stripped, and reprobed with total ERK-1 antibody (lower panel) to show equal loading.
