MAPK ERK Signaling Regulates the TGF-β1-Dependent Mosquito Response to Plasmodium falciparum
Figure 2
PD98059 inhibition of ERK phosphorylation increased TGF-β1-induced NOS expression in Anopheles cells.
(A) The MEK1/2 inhibitor PD98059 dose-dependently inhibited TGF-β1-dependent ERK phosphorylation in 4a3B cells. Cells were pre-treated with PD98059 for 40 min then treated with 60 pg/ml human TGF-β1 for 15 min. Cell lysates were analyzed by western blotting using anti-phospho-ERK or anti-ERK antibody. (B) PD98059 inhibited TGF-β1-dependent ERK phosphorylation in ASE cells. Cells were pre-treated with PD98059 at 4 or 10 µM for 40 min then treated with 6000 pg/ml TGF-β1 for 5 min. Cell lysates were subjected to western blot analysis as in (A). (C) Inhibition of ERK phosphorylation in 4a3B cells is persistent. 4a3B cells were pre-treated with 4 µM PD98059 or with an equivalent volume of dimethyl sulfoxide (DMSO; PD98059 diluent) then treated with 60 pg/ml TGF-β1 for 5 min to 6 h. Cell lysates were subjected to western blotting analysis as in (A). Immunoblots in A–C are representative of 2–3 independent experiments. (D) PD98059 reversed the inhibitory effect of ERK activation on TGF-β1 induced NOS expression. 4a3B cells were pre-treated with 4 µM PD98059 or an equivalent volume of DMSO, then treated with 60 pg/ml TGF-β1. An additional control group was treated with PBS (TGF-β1 diluent) at a volume equivalent to the TGF-β1 treatment. The data are represented as means±standard errors from three independent experiments. The data were analyzed using ANOVA for overall significance and by Student-Neuman-Keuls for multiple pairwise comparisons. Legend: * = p<0.05 (TGF-β1+PD98059 versus all other groups).