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Fig. 1. Necessity of Par-3 for the formation of AJs and TJs. (Aa) Reduction in the amount of Par-3 by RNAi in MDCK cells. The cell lysates were subjected to SDS-PAGE, followed by western blotting with the indicated Abs. (Ab) Reduction in the immunofluorescence signal for Par-3 at cell-cell adhesion sites between two Par-3-knockdown MDCK cells. After Ca²⁺ switch, the cells were fixed and stained with the anti-Par-3 pAb. (Ac) Reduction in the immunofluorescence signals for E-cadherin and occludin at cell-cell adhesion sites between two Par-3-knockdown MDCK cells. After Ca²⁺ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm. (Ba) Re-concentration of the immunofluorescence signals for E-cadherin and occludin at cell-cell adhesion sites in the Par-3-knockdown MDCK cells that re-expressed GFP–Par-3. After Ca²⁺ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm. (Bb) Exogenous expression of GFP–Par-3 in Par-3-knockdown MDCK cells. MDCK cells were co-transfected with pBS-H1-Par-3 and pEGFP-Par-3. The cell lysates were subjected to SDS-PAGE, followed by Western blotting with anti-Par-3 pAb. (Ca) Weak cell-cell adhesion activity of Par-3-knockdown MDCK cells. After Ca²⁺ switch, the wild-type MDCK cells transfected with pBS-H1-Par-3 or pBS-H1-scramble as a control were trypsinized in the presence of 1 mM CaCl₂ (TC treatment) or 1 mM EGTA (TE treatment) for 1 hour and dissociated through pipetting ten times. The extent of the cell dissociation was represented by the index N_TC/N_TE, where N_TC and N_TE were the total particle numbers after the TC and TE treatments, respectively. The results shown are representative of three independent experiments. (Cb) The paracellular diffusion of FITC-conjugated dextran (average 40 kDa) in Par-3-knockdown MDCK cells. Data are expressed as the means±s.d. of three independent experiments.

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