Figure 3. Linker-phosphorylated Smad2 (S255)–STAT3–p300 complex transactivates the Rorc and Il17a.

Interactions of endogenous proteins in T_H17 cells and exogenous proteins in 293T cells were determined by PLA. PLA signals (a–c,f) were quantified using the BlobFinder software (scale bars, 10 μm; nucleus, black; cytoplasm, white, n=10 fields). (a) Endogenous interaction between Smad2 and STAT3 in T_H17 cells. (b) Effects of truncated mutations in Smad2 on the interaction with STAT3 in 293T cells. (c) Effects of linker domain variations in Smad2 on the interaction with STAT3 in 293T cells. (d) Effects of Smad2 (S255A) on STAT3-induced activation of the Rorc promoter and the Il17a promoter constructs transfected in T_H17 cells were analysed by luciferase assay. (e) Flow cytometry analyses of IL-17A⁺RORγt⁺CD4⁺ T cells transduced with the indicated DNA constructs using Nucleofector (n=2). (f) Endogenous interactions between p300 and Smad2 or STAT3 in T_H17 cells were determined using PLA. (g) Effects of p300 on Smad2/STAT3-induced activation of the Rorc promoter (white) and the Il17a promoter (black) constructs transfected in 293T cells were analysed by luciferase assay. (h) Binding of Smad2 (white) and p300 (black) to the proximal promoter regions of the Rorc gene and the Il17a gene in T_H17 cells was determined using ChIP. ChIP data are shown as differential occupancy fold changes. Data are from one experiment representative of six (a), three (b–d) or two (e–h) independent experiments. Each experiment (d,g,h) was performed in triplicate (n=3). Data are mean+s.d.