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. 2022 Jun 14;13:3419. doi: 10.1038/s41467-022-31141-6

Fig. 4. UDCA promotes TGF-β1 ubiquitination at K315 by CHIP.

Fig. 4

a IB analysis of K48-linked, K63-linked and total ubiquitination of TGF-β1 in naïve CD4+ T cells stimulated with anti-CD3, anti-CD28, 50 μM UDCA and 1 nM Baf-A1 for 24 h. IB analysis was conducted after IP with anti-TGF-β1. b IB analysis of CHIP and TGF-β1 in naïve CD4+ T cells stimulated with anti-CD3 and anti-CD28 for 24 h. IB analysis was conducted after IP with anti-TGF-β1 or IgG. c–e IB analysis of TGF-β1 (c), FC analysis of Foxp3+CD4+ cells (d) and immunofluorescence analysis of LC3B and TGF-β1. Scale bar, 2 μm (e) in CHIP-knockdown naïve CD4+ T cells stimulated with anti-CD3, anti-CD28 and 50 μM UDCA for 24 h (c, e) or for 4 days (d). f, g IB analysis of TGF-β1 ubiquitination in the presence of E1, E2, Ub, purified CHIP, and TGF-β1 (f) or TGF-β1K315R (g). h–k Immunofluorescence analysis of p62 and TGF-β1K315R (h, j) or LC3B and TGF-β1K315R (i, k) in NIH-3T3 cells treated with 50 μM UDCA (h, i) or transfected with vectors expressing PRKACA (j, k) and transfected with the TGF-β1K315R mutant plasmid for 24 h. Scale bar, 2 μm. Representative results from two (e, h–k) or three (a–d, f, g) independent experiments are shown [n = 3 in (d); n = 8 in (e); n = 9 (DMSO) or 8 (UDCA) in (h); n = 9 (DMSO) or 10 (UDCA) in (i); n = 12 (Ctrl) or 8 (PRKACA) in (j); n = 11 in (k)]. **P < 0.01; ***P < 0.001; ns, not significant (unpaired two-tailed Student’s t test; mean and s.d.). See Source Data file for the exact P-values.