Figure 1.

Constitutive proteasomal degradation of steady-state Smad3 is regulated by Axin. (A) HaCaT cells were pretreated with 10 μM SB-431542 for 6 h, and 50 μg/mL CHX or 25 μM MG-132 was then added as indicated. Total cell lysates were probed for endogenous Smad3 and Smad2. γ-Tubulin was used as a loading control. (B) A ubiquitination assay of endogenous Smad3. HaCaT cells were pretreated with either vehicle (ethanol) or 10 μM SB-431542 for 1 h before the addition of 30 μM MG-132. After another 3 h, cells were lysed in SDS lysis buffer for immunoprecipitation (IP) with no antibody (−) or anti-Smad3 (Zymed). (C) Retrovirally infected stable populations of SNU475 cells expressing vector control or wild-type hAxin were pretreated with 10 μM SB-431542 and then treated with 50 μg/mL CHX for the indicated lengths of time. Endogenous proteins were probed. (D) Quantification of endogenous Smad3 level in C and in Alexander and DLD-1 cells tested in similar experiments with the presence of SB-431542. The level of Smad3 in each cell line at time “0” was set as 1.0. (E) Effective knockdown of endogenous Axin in 293T cells by two shRNAs designated as R1 and R2. pSuper-GFP was used as a nontargeting shRNA control. (F,G) Smad3 ubiquitination assays in SNU475 stable lines (F) and in 293T cells (G). pSuper-GFP was used as a control for Axin RNAi (R1 + R2). Cells were pretreated with 10 μM SB-431542 for 1 h followed by MG-132 treatment (25 μM) for 4 h and then lysed in RIPA buffer. Note in G that endogenous hAxin migrates faster than myc-mAxin due to smaller size.