Fig. 4.

Activation of p38 by contact disassembly regulates nuclear accumulation of MRTF and the SMA promoter. (A) Phospho-p38 was detected in lysates from confluent cultures treated with LCM for the indicated times. (B) Cultures were pretreated with DMSO or 10 μM SB203580 for 30 min prior to incubation with SFM or LCM for 1 h. Phospho-Hsp27 was detected by Western blotting. Membranes were reprobed for Hsp27. (C) The pSMA-Luc/pRL-TK reporter system was either transfected alone or with DN-p38, and the cells were pretreated with 10 μM SB203580 before Ca²⁺ removal (24 h). (D) Cells were treated as in B and the MRTF content of nuclear extracts was detected by Western blotting. Membranes were reprobed for histones. (E) Western blots in D were quantified by densitometry. Nuclear MRTF from LCM-treated cells corresponds to 100% (n = 3). (F) Nuclear translocation of MRTF upon Ca²⁺ removal (1 h, upper row) or scratch-wounding of the monolayer with a pipette tip (6 h, lower row) was followed in control and SB203580-pretreated samples by immunostaining. (G) LCM caused retarded migration of MRTF in total cell lysates (arrows), which was prevented by SB203580.