FIGURE 3.

Distribution and expression of Smads 1/5/8 during ATP depletion and recovery. A, LLC-PK₁ cells were cultured in normal growth medium or ATP depletion medium for 2 h or allowed to recover for an additional 2 or 4 h. Immunocytochemical localization of HuR, total R-Smads, and pSmad 1/5 was determined. B, total R-Smad, Smad 1/5/8, and pSmad 1/5 were assayed by Western analysis. β-Actin was included as a loading control. Bands from multiple Western blots as shown were quantified and expressed in graphical form. C, gel mobility shift assays and ChIP were performed on nuclear extracts from untreated (unt) or ATP-depleted/recovered (rec) LLC-PK₁ cells. In gel mobility shift assays (top), one major band (designated by the arrow) was found to specifically bind Smad 1/5/8 consensus motifs in the porcine HuR 5′-UTR. The intensity of this band increased following ATP depletion/recovery. In ChIP assays (bottom panel), antibody against Smad 1/5/8 precipitated DNA corresponding to the HuR 5′-UTR. D, introduction of exogenous Smad 1 into LLC-PK₁ cells increased HuR mRNA expression, as determined by competitive RT-PCR. An empty vector control produced no effect. The asterisks indicate p < 0.005, as determined by Student's t test.