Figure 3: Knockdown of A20 sustains TGF-β1-mediated TRAF6 polyubiquitination.

(a) A20, but not CYLD, is required for Smad6-mediated inhibition of TRAF6 polyubiquitination. HEK293 cells were reverse-transfected with 30 nM of control siRNA (siCON) or two independent A20 siRNAs (siA20#1 or #3) or CYLD siRNAs (siCYLD #2 or #3), respectively. After 24 h, Flag-TRAF6 and His-Ub plasmids were co-transfected with HA-SMAD6 into HEK293 cells in the indicated combinations. Cells were lysed in 6 M guanidine-HCl, and His-ubiquitinated proteins precipitated using Ni-NTA agarose beads. Ubiquitinated TRAF6 was observed by IB using anti-Flag antibody. (b–d) AML-12 cells (b,c) and mouse primary hepatocytes (d) were infected with lentiviruses expressing the indicated shRNAs targeting GFP (shGFP; negative control ), A20, or CYLD, and treated with 5 ng ml⁻¹ TGF-β1 for the indicated times. For (b–d), endogenous TRAF6 ubiquitination was observed by IP under denaturing conditions with anti-TRAF6 antibody and immunoblotted with anti-ubiquitin-HRP. Total cell lysates were immunoblotted with indicated antibodies. Expression of β-actin was used as a loading control. The data in (a–d) are representative of at least three independent experiments.