FIG. 3.
Effect of Rho GTPase on junctional disruption. (A) Cells were pretreated with PKA inhibitor (10−5 M) for 30 min, Epac, or nontargeting (Nt) siRNA for 1 day before 8-Bromo cAMP (10−5 M) treatment for 30 min and were then loaded with affinity precipitation in the presence of 10 μg of GST on glutathione-sepharose beads. After each binding reaction at 4°C, the proteins bound to the beads were separated by 15% SDS-PAGE and were then examined for GTP-bound Rac1 or Cdc42. The right panel of Fig. 2A depicted by bars denotes mean ± SE of three experiments for each condition determined by densitometry relative to each total of proteins. *P < 0.05 versus control. **P < 0.05 versus 8-Bromo cAMP. (B) Cells were transfected with Rac1 and Cdc42 siRNA (20 nM) or Nt siRNA for 1 day before treatment of 8-Bromo cAMP for 2 h and expression of phosphor-MLC, MLCK, and MLC was detected by western blotting. The right panel depicted by bars denotes mean ± SE of three experiments for each condition determined by densitometry relative to MLC for phospho-MLC and β-actin for MLCK. *P < 0.05 versus control. **P < 0.05 versus 8-Bromo cAMP alone. (C) The cells were pretreated with ML-7 (MLCK inhibitor, 10−5 M) or calyculin A (Cal A, 10−6 M, phosphatase inhibitor) before 8-Bromo cAMP (10−5 M) for 2 h. Complexes were immunoprecipitated from cell lysate with an anti-ZO-1 antibody and blotted with an antibody against Cx43, claudin, occludin, or E-cadherin. (D) Cells were incubated with lysosomal degradation inhibitor (chloroquine, 10−6 M) and proteasome inhibitor (lactacystin, 10−6 M) for 30 min before treatment of 8-Bromo cAMP for 2 h and expression of Cx43, claudin, occludin, E-cadherin, or ZO-1 in membrane and cytosol fraction, which was detected by western blotting. The right panels of C, D depicted by bars denote mean ± SE of three experiments for each condition determined by densitometry. *P < 0.05 versus control. **P < 0.05 versus 8-Bromo cAMP alone. (E) Oris™ cell migration assay was performed and stained with calcein-AM (5 μM). Fluorescence in the analytical zone was quantified with a plate reader. The example shown is a representative of five independent experiments. *P < 0.05 versus control. **P < 0.05 versus 8-Bromo cAMP alone. (F) Wound healing migration assay was performed and images acquired before and 24 h after 8-Bromo cAMP treatment. The right panel depicted by bars denotes mean ± SE of three experiments for each condition analyzed by Meta Morph v.7.01 software. The example shown is representative of three independent experiments. *P < 0.05 versus control. **P < 0.05 versus 8-Bromo cAMP alone.