Fig. 7. The post-degradation increase in the endogenous HEF1 protein level is due to an increase in HEF1 mRNA and elevated HEF1 protein level is inhibitory to Smad3-mediated gene responses of TGF-β. (A) TGF-β induces a rapid increase in HEF1 mRNA level in A549 cells. Cells were exposed to 400 pM TGF-β and mRNA was isolated and subjected to northern blotting using ³²P-labeled HEF1 as a probe (top panel). The 5.4 and 3.8 kb HEF1 transcripts are indicated. The GAPDH level was used to monitor the total amount of mRNA in each lane (bottom panel). (B) Increased HEF1 protein level is inhibitory to TGF-β-induced activation of the Smad3-responsive reporter CAGA-luc. A549 cells were transiently transfected in duplicate with CAGA-luc (0.15 µg) and the internal control CMV-RL Renilla (0.05 µg) in the absence or presence of HEF1 expression vector (0.3 µg), as indicated. The following day, cells were either untreated or treated with 100 pM TGF-β in the absence or presence of 50 µM LLnL for 9 h before cells were harvested for luciferase assays.