Fig. 5: The binding of TGF-β1 phosphorylated at T282 by PKA to CHIP is increased.
From: Ursodeoxycholic acid reduces antitumor immunosuppression by inducing CHIP-mediated TGF-β degradation
a IB analysis of Ser/Thr-phosphorylated TGF-β1 in naïve CD4+ T cells stimulated with anti-CD3, anti-CD28 and 50 μM UDCA and treated with or without 2 μM H89 for 24 h. IB analysis was conducted after IP with anti-TGF-β1. b IB analysis of assay mixtures containing Flag-TGF-β1, Myc-PRKACA and ATP after reaction at 30 °C for 30 min in vitro. c IB analysis of CHIP and TGF-β1 in naïve CD4+ T cells stimulated with anti-CD3, anti-CD28 and 50 μM UDCA and treated with or without 2 μM H89 for 24 h. IB analysis was conducted after IP with anti-TGF-β1. d IB analysis of Ser/Thr-phosphorylated TGF-β1 in HEK293T cells transfected with vectors expressing Flag-TGF-β1WT, Flag-TGF-β1T282A, and Myc-PRKACA for 24 h. e IB analysis of assay mixtures containing Flag-TGF-β1 or Flag-TGF-β1T282A plus Myc-PRKACA and ATP after reaction at 30 °C for 30 min in vitro. f IB analysis of TGF-β1 in the mixture of His-CHIP and Flag-TGF-β1WT and the mixture of Flag-TGF-β1T282A after PD with anti-His magnetic beads. g IB analysis of TGF-β1 ubiquitination in the presence of E1, E2, Ub, purified CHIP and TGF-β1WT or TGF-β1T282A. h, i Immunofluorescence analysis of LC3B and TGF-β1 in HEK293T cells transfected with Flag-TGF-β1WT or Flag-TGF-β1T282A for 24 h. Scale bar, 2 μm. (h) FC analysis of Foxp3+CD4+ cells induced by culture with supernatants from these cells for 3 days (i). Representative results from two (h) or three (a–g, i) independent experiments are shown (n = 8 in h; n = 3 in i). *P < 0.05; **P < 0.01 (unpaired two-tailed Student’s t test; mean and s.d.). See Source Data file for the exact P-values.
