Extended Data Figure 3: SKI identification and its degradation upon low doses of TGFβ.

a, Schematic of quantitative immunoprecipitation and mass spectrometry proteomic strategy to identify SMAD4-binding proteins under different conditions. In one scheme, to identify SMAD4-binding protein in the absence of TGFβ signalling, CD4⁺ T cells from Cd4-cre;Smad4^fl/fl (S4 KO) mice were sorted and activated in the presence of IL-6 + inhibitor in the SILAC/AACT medium provided either with amino acids containing light (L) isotopes or with amino acids containing heavy (H) isotopes. Cells were then transduced with retroviruses expressing either Flag tag (RV Flag) or Flag tag and SMAD4 fusion protein (RV Flag-S4). Cells were harvested and mixed 4 days after activation. Immunoprecipitation was performed using anti-Flag. Immunoprecipitated proteins were processed and subjected to quantitative mass spectromtery analysis. Proteins with a heavy/light ratio of more than 2 were identified. In the other scheme, to identify the proteins whose SMAD4 interaction was perturbed upon TGFβ stimulation, CD4⁺ T cells from wild-type mice were sorted and activated either in the presence of IL-6 + inhibitor in the SILAC/AACT medium provided with amino acids containing light isotopes or in the presence of IL-6 + TGFβ in the SILAC/AACT medium provided with amino acids containing heavy isotopes. Cells were harvested and mixed 4 days after activation. Immunoprecipitation was performed using anti-SMAD4 antibody. Immunoprecipitated proteins were processed and subjected to quantitative mass spectrometry analysis. Proteins with a heavy/light ratio of less than 1 were identified. The commonly identified protein SKI in the two experiments was subjected to further investigation. b, CD4⁺ T cells from wild-type mice were activated in the presence of IL-6 and the indicated doses of TGFβ. Cells were harvested after 24 h. SKI protein expression was detected by immunoblotting. Results are representative of three experiments with similar results. c, CD4⁺ T cells from wild-type mice were activated in the presence of IL-6 and the indicated doses of TGFβ. IL-17A⁺ and Foxp3⁺ cells were assessed by flow cytometry on day 4. Representative results (top) and statistical analysis (bottom) of five experiments are shown (centres indicate mean values). d, CD4⁺ T cells from wild-type or Cd4-cre;Smad2^fl/fl (S2 KO) mice were activated in the presence of IL-6 for 24 h. Cells were then stimulated with the indicated conditions for an additional 1 h with or without 10 μM SIS3 (specific inhibitor of Smad3 phosphorylation). SKI protein expression and Smad3 phosphorylation were assessed by immunoblotting. Results are representative of three experiments with similar results.

Source data