Figure 3

Proteasome blockade partially antagonizes TGF-β activity and stabilizes SKI. A. 1205Lu cells were incubated for 1 h in the absence (DMSO) or presence of the proteasome inhibitor MG132 (10 μM) prior to addition of TGF-β (10 ng/ml). Protein extracts were prepared 60 min later and subjected to Western analysis for SKI content. B, C. 1205Lu (panel B) and Dauv-1 (panel C) melanoma cells were transfected in triplicate dishes with (CAGA)₉-MLP-luc and pRL-TK. 4 h later, cells were incubated with either DMSO, MG132 or ALLN 1 h prior to the addition of TGF-β (10 ng/ml). Reporter activity was measured 16 h later. Results are mean+/- SD from one representative experiment. D, E. 1205Lu melanoma cells were incubated with either DMSO or MG132 1 h prior to the addition of TGF-β (10 ng/ml). RNA was extracted 8 h later. IL-11 (panel D) and PTHrP (panel E) expression was measured by quantitative RT-PCR.