Fig. 7.

Strong hyperosmolarity promotes rapid proteasomal degradation of MRTF. A: LLC-PK1 cells were serum deprived for 3 h, followed by pretreatment with DMSO, the pan-caspase inhibitor zVAD-fmk (20 or 40 μM), or MG132 (25 μM) for 1 h. Cells were then exposed to iso- or hypertonic (+200 mM NaCl = 700 mosM total) treatment in serum-free medium for 2 h in the presence of the indicated inhibitors. Cell lysates were immunoblotted for endogenous MRTF, cleaved caspase-3, and GAPDH. B: densitometric analysis of the effect of zVAD-fmk and MG132 on endogenous MRTF protein expression under iso- and hypertonic conditions. In the top panel expression is normalized to the untreated isotonic samples, whereas the bottom panel shows the relative changes induced by hypertonicity, with the expression normalized to the isotonic level in each group. The difference between iso- and hypertonic samples is significant for the control and zVAD-fmk-treated samples (P < 0.01) and NS for MG132. C: LLC-PK1 cells were transfected with MRTF-hemagglutinin (HA) plasmid. After 24 h, cells were treated and processed as in A.