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Fig. 3. Demonstration of involvement of YWK-II protein in MIS-induced ERK1/2 phosphorylation coupled to G_o, G_o, Ras and PKC. Cells were lysed and assayed for p-ERK1/2 and total ERK1/2 by western blotting. The averaged ratio values of triplicate experiments are presented with corresponding western blot results. Unless stated otherwise, the cells were starved for 16 hours prior to stimulation with 0.35 nM rMIS at 37°C for 5 minutes. Statistical analysis was performed using Student's t-test (two-tailed comparison of means for paired samples). (A) Enhanced rMIS-induced ERK1/2 phosphorylation in YWK-II-overexpressing CHO cells (pEGFP-N1-YWK-II) as compared to that in EGFP-overexpressing CHO cells (pEGFP-N1) and non-transfected CHO cells. Different concentrations of rMIS used: 0, 0.035 and 0.35 nM (P<0.01). (B) Concentration-dependent effect of rMIS on ERK1/2 phosphorylation in EGFP-YWK-II-overexpressing CHO cells. Different concentrations of rMIS used: 0, 0.0175, 0.035, 0.35, 3.5 and 35 nM. (C) Effect of PTX on rMIS-induced ERK1/2 phosphorylation in EGFP-YWK-II-overexpressing CHO cells. Cells were starved for 16 hours and pretreated with PTX (1 µg/ml) or left untreated for 24 hours prior to stimulation with rMIS (P<0.01). (D) Effects of C-terminal regions of G_i1/2, G_o1 and G_o2 on rMIS-induced ERK1/2 phosphorylation. pcDNA3.1(+)-G_i1/2, pcDNA3.1(+)-G_o1 and pcDNA3.1(+)-G_o2 were transfected into EGFP-YWK-II-overexpressing CHO cells for 24 hours (P<0.05). (E) Effect of ARK1 on rMIS-induced ERK1/2 phosphorylation. The EGFP-YWK-II-overexpressing CHO cells were transfected with pCMV-ARK1-C for 24 hours (P<0.05). (F) Effects of pCMV-RasN17 and pCMV-RasV12 on rMIS-induced ERK1/2 phosphorylation. EGFP-YWK-II-overexpressing CHO cells were transfected with pCMV-RasN17 and pCMV-RasV12 for 24 hours (P<0.05). (G) Effect of GF109203X on rMIS-induced ERK1/2 phosphorylation in EGFP-YWK-II-overexpressing CHO cells. Cells were starved for 16 hours and pretreated with GF109203X (3.5 µM) or left untreated for 2 hours prior to stimulation with rMIS (P<0.01). (H) Effect of pCMV-RasN17 and GF109203X on rMIS-induced ERK1/2 phosphorylation. EGFP-YWK-II-overexpressing CHO cells were transfected with pCMV-RasN17 for 24 hours. The cells were starved for 16 hours and treated with or without GF109203X (3.5 µM) for 2 hours prior to stimulation with rMIS (P<0.01).

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