Fig. 7

Counteracting effects of DUSP3 and FAK regulates OCLN ubiquitination and degradation. A H1299-OCLN and OCLN-4YF cells were treated with cycloheximide (50 μg/ml) for indicated time periods and the cell lysates were subjected to immunoblot analyses. The arrowhead indicated the position of OCLN with slower mobility. B H1299-OCLN and OCLN-4YF cells were pre-treated with cycloheximide (50 μg/ml) for 30 min and then were treated with H₂O₂ (1 mM) for indicated periods. The cell extracts were then collected and subjected to immunoblot analyses. C H1299-OCLN and OCLN-4YF cells were pre-treated with MG132 (0 or 50 mM) for 1 h and then were treated with or without H₂O₂ (1 mM) for 1 h. The cell extracts were collected and subjected to immunoblot analyses. The graphs under the immunoblots showed the mean and standard deviations of three independent experiments (A-C). * p < 0.05, ** p < 0.01. D Cell extracts prepared from HA-ubiquitin (HA-Ub)-transfected H1299-OCLN and OCLN-4YF were subjected to immunoprecipitation of Flag-OCLN, and the immunocomplexes were then analyzed for the presence of indicated proteins. E and F H1299-OCLN and OCLN-4YF cells were treated with or without H₂O₂ (1 mM) for 30 min and were subjected to extract preparation. Flag-tagged OCLN and OCLN-4YF were immunoprecipitated and the immunocomplexes were then analyzed for the presence of indicated proteins. G H1299-DUSP3_TR/OCLN and H1299-DUSP3-CS_TR/OCLN cells were treated with or without Tet (1 mg/ml) for 12 h and then were treated with H₂O₂ (0.5 mM) for 30 min. Cell extracts were prepared and Flag-tagged OCLN and OCLN-4YF were immunoprecipitated. The immunocomplexes were then analyzed for the presence of indicated proteins. Cell extracts (2% percent input) were also examined for the relative amounts of individual proteins