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Fig. 1. Eph4 transfectants expressing GFP-claudin-3. (A) A membrane folding model for GFP-claudin-3 (NGFP-Cld3), in which GFP was fused to the N-terminus of mouse claudin-3. (B) Expression levels of endogenous claudin-3 (endogenous Cld3) and exogenous NGFP-Cld3 (GFP-Cld3). The whole cell lysate of parental Eph4 cells, as well as two independent Eph4 clones expressing NGFP-Cld3 (Eph4-GFP-Cld3#1, Eph4-GFP-Cld3#2), was immunoblotted with anti-claudin-3 pAb or anti-GFP pAb. In each lane, the same amount of total protein was applied. The amounts of endogenous claudin-3 (Q1) and NGFP-Cld3 (Q2) in two stable clones were quantified as described in Materials and Methods, and their Q2/Q1 ratios were calculated. (C) Colocalization of NGFP-Cld3 with endogenous claudin-4 and ZO-1 in Eph4:NGFP-Cld3 cells. Cells were stained with anti-claudin-4 mAb or anti-ZO-1 mAb. (D) Confirmation of the correct membrane folding of NGFP-Cld3 in Eph4:NGFP-Cld3 cells. In the presence (+) or absence (–) of Triton-X 100, cells were stained with anti-GFP pAb (left panels) or anti-claudin-3 pAb, which recognizes the C-terminal tail of claudin-3 (right panels). Bars: 10 µm (C); 10 µm (D).

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