Skip to main content
. 2011 Jul 27;286(37):32502–32512. doi: 10.1074/jbc.M111.227561

FIGURE 3.

FIGURE 3.

RB1CC1 is a novel positive regulator of TGF-β signaling. A, a luciferase reporter assay was performed in HEK293 cells using (CAGA)9-MLP-Luc2. Cells were stimulated by co-expression of a constitutively active TGF-β type I receptor (ALK-5-TD). Error bars represent S.D. B, knockdown of RB1CC1 using siRNA oligos is shown. HaCaT cells were transfected with siRNA oligos and harvested 48 h later. Expression of RB1CC1 protein in cell lysates was examined by immunoblotting. 80 μg of protein was applied on each lane. NC, control siRNA. C, knockdown of RB1CC1 down-regulates Smad signaling. siRNA oligos were transfected into HaCaT cells 1 day before transfection of luciferase reporter plasmids. Cells were then stimulated with TGF-β, and luciferase activities were measured. Error bars represent S.D. D, shown is the effect of RB1CC1 knockdown on TGF-β-responsive gene expression in HaCaT cells. Cells were transfected with control or RB1CC1 siRNA #2. Forty-eight hours later cells were stimulated with TGF-β3 (1 ng/ml) and were harvested at 1.5, 6, or 24 h after stimulation. Induction of target genes was examined by quantitative real-time-PCR analysis and normalized by GAPDH expression levels. Expression of c-Ski was also examined as a control. Error bars represent S.D. E, RB1CC1 siRNAs inhibit the cytostatic effect of TGF-β. HaCaT cells were transfected with siRNAs and stimulated with TGF-β 36 h later. [3H]Thymidine incorporation was measured the next day. The obtained data were normalized to the incorporation without TGF-β. Error bars represent S.D. F, RB1CC1 enhances Smad signaling via its C-terminal region. HEK293 cells were transfected with deletion mutants of RB1CC1, (CAGA)9-MLP-Luc2 reporter, and ALK-5-TD. Error bars represent S.D. The lower panel shows expression of RB1CC1 deletion mutants. G, RB1CC1 fails to enhance TGF-β signaling upon knockdown of Arkadia. Luciferase reporter assay was performed using HEK293 cells transfected with control siRNA or Arkadia siRNA. Error bars represent S.D. *, p < 0.01; n.s., not significant.

HHS Vulnerability Disclosure