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. 2021 Oct 21;297(6):101330. doi: 10.1016/j.jbc.2021.101330

Figure 6.

Figure 6

IL-6 receptor signaling blocks SMAD3/4 activation of PKA during Th17 induction.A, primary murine CD4+ T cells isolated by negative selection were activated with different combinations of TCR/CD28, TGF-β, and IL-6 signaling inputs for 10 min. SMAD3 was immunoprecipitated. Immunoblotting was performed for SMAD3, STAT3, SMAD4, and PKA. Densitometry was performed on immunoblots to determine the relative amount of (B) SMAD4, (C) STAT3, and (D) PKA in the SMAD3 IP across three biological replicates. Shown are mean ± SD; p values were calculated by one-way ANOVA. E, primary murine CD4+ T cells were activated for 10 min with constant amounts of plate bound anti-CD3 antibody, soluble anti-CD28 antibody, and TGF-β with varying amounts of IL-6. SMAD3 was immunoprecipitated and immunoblotting was performed to monitor the amount of PKA that coprecipitated with SMAD3. This experiment corresponds to the data presented in Figure 5B and the SMAD3 immunoblots are therefore identical between both panels. F, densitometry was performed on immunoblots from panel F across three biological replicates where PKA was normalized to SMAD3 in the IP. G, primary murine CD4+ T cells activated under Treg (plate bound anti-CD3 antibody, soluble anti-CD28 antibody, and TGF-β) or Th17 (plate bound anti-CD3 antibody, soluble anti-CD28 antibody, TGF-β, and IL-6) stimulation for various time points. Immunoblotting for p-PKA (T197) and total PKA protein were performed. H, densitometry was performed on immunoblots from panel G across three biological replicates. Shown are mean ± SD; p values were calculated by two-way ANOVA (∗∗∗∗p < 0.0001). I, CD4+ T cells were activated under TH17 or Treg conditions for 10 min in the presence or absence of 200 nM JAK2 inhibitor. Cell lysates were immunoblotted for p-PKA (T197) and total PKA protein. J, densitometry was performed on immunoblots from panel I where p-PKA (T197) was normalized to total PKA protein across three independent experiments. Shown are mean ± SD; p values were calculated by one-way ANOVA. Source data are provided for the immunoblots in panels A, E, G and I.