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Fig. 9. Involvement of Par-3 in the trans-interacting E-cadherin-induced, but not nectin-induced, activation of Rac. (A) Reduction in the activation of Rac1 in Par-3-knockdown MDCK cells. After Ca²⁺ switch, the cell lysates were subjected to pull-down assay using GST-PAK-CRIB, followed by western blotting using the anti-Rac1 mAb. The results shown are representative of three independent experiments. (B) No effect of Par-3 knockdown on nectin-induced cell-spreading. The nectin-1-MDCK cells co-transfected either with pBS-H1-Par-3 and pEGFP or with pBS-H1-scramble and pEGFP as a control were cultured on the Nef-3- or IgG-coated coverslips for 2 hours. The cells were fixed and stained for F-actin with rhodamine-phalloidin. Bars, 10 µm. Bars in the graph represent the percentage of spreading cells with lamellipodial cell protrusions of the 100 GFP-positive cells counted, and are expressed as means±s.d. of three independent experiments. (C) Reduction of E-cadherin-induced cell-spreading by Par-3 knockdown. The E-cadherin-MDCK cells co-transfected either with pBS-H1-Par-3 and pDsRed, with pBS-H1-Par-3, pEGFP-V12Rac1, and pDsRed, or with pBS-H1-scramble and pDsRed as a control were cultured on the Cef- or IgG-coated coverslips for 2 hours. The cells were fixed and stained for F-actin with Cy5-phalloidin. Bars, 10 µm. Bars in the graph represent the percentage of spreading cells with lamellipodial cell protrusions of the 100 DsRed-positive cells counted, and are expressed as means±s.d. of three independent experiments. (D) No effect of afadin knockdown on E-cadherin-induced cell-spreading. The E-cadherin-MDCK cells co-transfected either with pBS-H1-afadin and pDsRed or with pBS-H1-scramble and pDsRed as a control were cultured on the Cef- or IgG-coated coverslips for 2 hours. The cells were fixed and stained for F-actin with Cy5-phalloidin. Bars, 10 µm. Bars in the graph represent the percentage of spreading cells with lamellipodial cell protrusions of the 100 DsRed-positive cells counted, and are expressed as means±s.d. of three independent experiments. (Ea) Reduction in the immunofluorescence signals for E-cadherin and occludin, but not that for GFP-V12Rac1, at cell-cell adhesion sites in the Par-3-knockdown MDCK cells that expressed GFP-V12Rac1. After Ca²⁺ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm. (Eb) Reduction in the immunofluorescence signals for E-cadherin and occludin, but not that for GFP-V12Rac1 and FLAG-afadin, at cell-cell adhesion sites in the Par-3-knockdown MDCK cells that expressed GFP-V12Rac1 and FLAG-afadin. After Ca²⁺ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm.

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