Figure 5. ERK induces Smad2 linker phosphorylation that facilitates T_H17 differentiation.

Purified CD4⁺ T cells were activated under T_H17-polarizing condition with the indicated doses of TGF-β1 and plate-coated anti-CD3 antibody, or small molecule inhibitors (EW-7197, ALK5 inhibitor; PD98059, MEK inhibitor) for 3 days. (a) Flow cytometry analyses of IL-17A⁺RORγt⁺CD4⁺ T cells treated with TGF-β1 and plate-coated anti-CD3 antibody. (b) Endogenous expression of pSmad2L in T_H17 cells treated with TGF-β1 and plate-coated anti-CD3 antibody was determined using PLA. (c) Flow cytometry analyses of phospho-ERK in T_H17 cells treated with TGF-β1 and plate-coated anti-CD3 antibody. (d) Flow cytometry analyses of IL-17A⁺RORγt⁺CD4⁺ T cells treated with EW-7197. (e) Endogenous expression of pSmad2L in T_H17 cells treated with EW-7197 was determined using PLA. (f) Flow cytometry analyses of phospho-ERK in T_H17 cells treated with EW-7197. (g) Flow cytometry analyses of IL-17A⁺RORγt⁺CD4⁺ T cells treated with PD98059. (h) Endogenous expression of pSmad2L in T_H17 cells treated with PD98059 was determined using PLA. The values of the mean fluorescence intensity (MFI) are shown in graphs. PLA signals (b,e,h) were quantified using the BlobFinder software (scale bars, 10 μm; nucleus, black; cytoplasm, white, n=10 fields). Data are representative of two (a–h) independent experiments. Data are mean+s.d.