Figure 2: Smad6 downregulates TRAF6 polyubiquitination.

(a) Smad6 inhibits TRAF6 polyubiquitination in a dose-dependent manner. Plasmids encoding Flag-TRAF6 and His-Ub were co-transfected with increasing amounts of HA-SMAD6 plasmid into HEK293 cells. (b) Plasmids encoding wild-type or lysine mutants (K48 and K63) of His-Ub were co-transfected with Flag-TRAF6 and HA-SMAD6 plasmids into HEK293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 h, and Ni-NTA-mediated pull-down assays were performed. (c) Plasmid encoding Flag-SMAD6 was transfected into AML-12 cells and treated with TGF-β1 for 30 min. After IP against endogenous TRAF6, K63-linked polyubiquitination of TRAF6 was observed by IB using a K63-specific ubiquitin antibody. (d) Plasmids encoding Flag-TRAF6 and His-Ub were co-transfected with HA-SMAD6, HA-SMAD7, and HA-A20 plasmids, respectively. (e) Smad6 specifically inhibits TRAF6 ubiquitination, but not TAK1. Plasmids encoding Flag-TRAF6, Myc-TAK1, His-Ub, and HA-SMAD6 were co-transfected into HEK293 cells in the indicated combinations. The ubiquitinated proteins and total cell lysates were observed by Ni-NTA-mediated pull-down assay and IB using the indicated antibodies. (f) AML-12 cells were infected with lentiviruses expressing shRNAs targeting GFP (shGFP; negative control ) or shSMAD6#4 and treated with 5 ng ml⁻¹ TGF-β1 for the indicated times. Endogenous TAK1 ubiquitination was observed by immunoprecipitation under denaturing conditions with an anti-TAK1 antibody and immunoblotted with anti-ubiquitin-HRP. Total cell lysates were immunoblotted with indicated antibodies. Expression of the β-actin was used as a loading control. All data are representative of at least three independent experiments.