Fig. 6

MICAL2 depletion leads to reduced E-cadherin degradation. A BGC-823 cells transfected with control siRNA or siRNA targeting MICAL2 (siMICAL2#2) were treated with 10 μg/mL cycloheximide (CHX, an inhibitor of protein synthesis) following which the cells were lysed and the E-cadherin protein level was determined by western blotting. GAPDH was used as the loading control. B BGC-823 cells transfected with siMICAL2 were treated with chloroquine (10 μM) or MG-132 (20 μM) for 24 h. Total proteins were then extracted from the lysates and subjected to western blotting for the measurement of E-cadherin expression levels. GAPDH served as the loading control. C BGC-823 cells were co-transfected with HA-ubiquitin and siMICAL2, following which E-cadherin ubiquitination was assayed. D Co-immunoprecipitation assays were used to detect the binding of endogenous E-cadherin to β-catenin in cells transfected with siMICAL2. E Representative immunofluorescence micrographs of BGC-823 cells stained for E-cadherin and β-catenin. Scale bar, 10 μm. **P < 0.01, ***P < 0.001