Fig. 5. Regulation of E-cadherin-catenin complex by Src42A. (A) Complex formation by E-cadherin, Arm and Src42A. Expression constructs of E-cadherin-GFP and Src42A were transfected into S2 cells and the E-cadherin-Arm complex was analyzed by immunoprecipitation. Blue and red boxes mark the lanes of whole cell extract and immunoprecipitates of anti-Arm, respectively. The expression of E-cadherin-GFP stabilized Arm in apparent high and low molecular weight forms, indicated by H and L. Although both Src42A^ACT and Src42A^DN in whole cell extract were phosphorylated at Y400, Src42A^DN in complex with Arm was under phosphorylated. (B) Src42A downregulates the cell surface level of E-cadherin. Surface biotinylation was applied to S2 cells expressing E-cadherin-GFP and Src42A. Western blot analyses were performed on whole cell extracts (whole) and proteins were recovered by avidin beads (surface). Septin and avidin-reacting proteins were used as loading controls for each blot. The positions of marker proteins are shown in the avidin panel. (C) Downregulation of E-cadherin-GFP by Src42A^ACT. Transfected S2 cells were stained with a method to detect extracellular E-cadherin (surface, magenta). GFP signal in the intracellular domain (intra, green) is shown. Src42A^ACT reduced both signals. (D) The amount of E-cadherin was reduced in Src mutant embryos. Control and Src42A^myr; Src64B^P1 embryos (src^-/-) were collected and analyzed for E-cadherin and pSrc expression by western blotting. Protein extracts were analyzed in the same gels and blots. Relative amount is shown above each band.