Fig. 5

Smad6 negatively regulates PIAS3 via the ubiquitin-proteasome pathway. a Nuclear-Smad6 OE decreased endogenous PIAS3 levels in A172 cells through the ubiquitin-proteasome pathway. MG132 (10 μM) or Ub inhibitor (20 ng ml⁻¹) was added 12 h before collecting. b Nuclear-Smad6 decreased exogenous PIAS3 through the ubiquitin-proteasome pathway. HA-PIAS3 plasmid (600 ng) was co-transfected with increasing amounts of nuclear-Smad6 construct (0, 0.2, 1.0 μg per well in a 6-well plate) into 293T cells. MG132 or Ub inhibitor was added 12 h before harvest. c,d Nuclear-Smad6-mediated ubiquitination and proteasomal degradation of endogenous and of exogenous PIAS3. After infected (c) or transfected (d) with indicated constructs for 36 h, cells were treated with 10 μM MG132 for 4 h before collecting. Immunoprecipitation (IP) was performed using the indicated antibodies