Fig. 4. Dissociating epithelial junctions regulate SRF via MAL. (A) Effects of MAL constructs or the F-actin stabilising mutant actin G15S. 0.4 µg MAL (f.l.), 2.5 µg MAL NB1, 2.5 µg MAL NC, 0.4 µg MAL N (scheme) or 2.5 µg actin G15S were co-transfected and Ca²⁺ exchange was carried out as in Fig. 3A. Luciferase activity is normalised to pRL-TK, with error bars indicating s.e.m. (n=3). (B) Dissociation of the actin-MAL complex upon Ca²⁺ withdrawal. MDCK cells were electroporated with 10 µg Flag-actin and 10 µg MAL-HA. 36 hours later, the medium was exchanged to low Ca²⁺ for the times indicated and cell lysates were immunoprecipitated with anti Flag. Proteins were visualised using antibodies against Flag and HA-tags. Latrunculin B (3 µM) or cytochalasin D (10 µM) was added 30 minutes before medium exchange. Relative association was calculated from densitometric analysis. Mock, medium exchange with 1.8 mM Ca²⁺; –Ca²⁺, medium exchange with 0.02 mM Ca²⁺.