FIGURE 5.

Increase of cell proliferation and phospho-ERK expression by TGFβ2 in SH-SY5Y cells overexpressing RFX1. A, cells were cultured in medium without fetal bovine serum for 24 h and then with 20% fetal bovine serum in the presence of 2 or 20 ng/ml of TGFβ2 for 2 days. Cell numbers were then measured and normalized by those without exogenous TGFβ2. B–E, cells were treated identically as described for panel A and then harvested for Western blotting of RFX1, phospho-ERK (pERK), total ERK (tERK), phospho-SMAD2/3 (pSMAD2/3), total SMAD2/3 (tSMAD2/3), and phospho-SMAD1/5/8 (pSMAD1/5/8). The RFX1, ERK, and SMAD Western blot results were normalized by those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The RFX1, ERK, and SMAD results from cells incubated with exogenous TGFβ2 were then normalized by those from cells without exogenous TGFβ2. F, cells overexpressing RFX1 were cultured in medium without fetal bovine serum for 24 h and then with 20% fetal bovine serum in the presence of 0.5 μm PD173074 with or without 20 ng/ml of TGFβ2 for 2 days. The expression of pERK and tERK in these cells was quantified by Western blotting. The normalization process was identical to that described for panels B–E. Results are mean ± S.D. (n = 3–4). *, p < 0.05 compared with the corresponding results without exogenous TGFβ2.