Skip to main content
. 2005 Jul 1;33(12):3708–3721. doi: 10.1093/nar/gki679

Figure 2.

Figure 2

Functional properties of a Smad3 mutant lacking part of the middle region. (A) Schematic representation of wild-type Smad3 and the Smad3 Δ200-230 mutant, tagged with a 6myc epitope at their N-terminus. Vertical dashed lines and numbers show the coordinates of the internal deletion. (B) HEK293T cells were transiently transfected with expression vectors for wild-type Smad3 or the Smad3 Δ200–230 mutant, and the expression levels of Smad3 proteins were monitored by immunoblotting using an anti-myc monoclonal antibody. (C) HEK293T cells were transiently transfected with expression vectors for wild-type Smad3 or the Smad3 Δ200–230 mutant. The transfected cells were treated with the protein synthesis inhibitor cycloheximide (50 μg/ml) for various time periods, and the expression levels of Smad3 proteins were monitored by immunoblotting using an anti-myc monoclonal antibody. Relative total protein levels were estimated by monitoring the expression of endogenous α-tubulin. The position of the 6myc-Smad3 proteins and α-tubulin is shown with arrows. The asterisk shows a 6myc-tagged Smad3 (Δ200–230) mutant with a lower electrophoretic mobility that is possibly derived from the utilization of an alternative translation termination signal. (D) JEG-3 (Smad3 −/−) choriocarcinoma cells were transfected with expression vectors for 6xmyc-Smad3 or 6xmyc-Smad3 (Δ200–230) along with the p(CAGA)12-E1B-Luc reporter, in the absence or in the presence of an expression vector for ALK5-ca. The normalized mean values of luciferase activity (±SEM) are shown with a bar graph. (E) Breast cancer MDA-MB-468 (Smad4 −/−) cells were transfected with expression vectors for 6xmyc-Smad3 or 6xmyc-Smad3 (Δ200–230) along with the p(CAGA)12-E1B-Luc reporter in the absence or in the presence of an expression vector for 6myc-Smad4 and TGFβ (200 pM). The normalized mean values of luciferase activity (±SEM) are shown with a bar graph. (F and G) Human hepatoma HepG2 cells were transfected with expression vectors for 6xmyc-Smad3 or 6xmyc-Smad3 (Δ200–230) in the absence or in the presence of an expression vector for 6myc-Smad4 and ALK5-ca and luciferase reporter plasmids bearing the promoters of the mouse Smad7 gene (−4200/+110) (F) or the human PAI-1 gene (−800/+71) (G) as indicated. The normalized mean values of luciferase activity (±SEM) are shown with bar graphs.