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. 2013 Feb 18;8(2):e56585. doi: 10.1371/journal.pone.0056585

Figure 6. TAZ enhances TGF-β-mediated Smad nuclear accumulation.

Figure 6

(A) The UAMS-32 cells were transfected with pEF-BOS (BOS) or pEF-Flag-TAZ (TAZ). Endogenous Smad2/3 and Smad4 localization was determined following treatment with the vehicle or TGF-β. (B) Sub-cellular localization of Smad2/3 and Smad4. UAMS-32 cells were transiently transfected with pEF-BOS or pEF-Flag-TAZ, fractionated into nuclear and cytosolic fractions, and analyzed by immunoblotting for Smad2/3, Smad4, and Flag. The purity of nuclear and cytosolic fractions was confirmed by immunoblotting for Calnexin. (C) C3H10T1/2 cells transfected with pEF-BOS or pEF-Flag-TAZ were cultured for 8 days in osteogenic medium (OM) including ascorbic acid and β-glycerophosphate in the absence or presence of TGF-β inhibitors (SB-431542 or SB-525334). ALP activity was measured in the cell layer and normalized to cellular protein content. Data are expressed as means ± SD (* p<0.05 vs. pEF-BOS, # p<0.01 vs. vehicle) (D) Quantitative RT-PCR analysis of Col 1 and ALP in C3H10T1/2 cells (* p<0.05 vs. pEF-BOS, # p<0.01 vs. vehicle).