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Fig. 1. N-cadherin/ER-LBD construct and its expression in L cells. (A) Full-length human N-cadherin (Ncad) cDNA fused at the 3' end to a modified estrogen receptor ligand-binding domain (ER-LBD) that binds 4-hydroxytamoxifen (4OHT) but not 17ß-estradiol. Pre and pro regions of the N-cadherin protein are cleaved off in cells to generate the mature N-cadherin. tm, transmembrane domain; cyto, cytoplasmic domain. (B) Western immunoblot analysis of N-cadherin, NcadER, 
-catenin, ß-catenin and p120ctn with GAPDH as a control. L cells stably expressing the N-cadherin/ER-LBD construct (LNER) were treated with 100 nM 4OHT (+) or vehicle (-) for 5 hours. Non-transfected L cells (L) and L cells expressing wild-type N-cadherin (LN), in the absence of 4OHT (-), served as negative and positive controls, respectively. Cells were extracted with 0.5% NP40 and soluble proteins resolved by SDS-PAGE, transblotted electrophoretically to nitrocellulose and immunoblotted with antibodies to N-cadherin, the three catenins, and GAPDH. The parent L cells lacked endogenous N-cadherin, and - and ß-catenin were not detected in these cells as the proteins are degraded in the absence of a cadherin. Similar levels of NcadER (molecular weight 
150 kDa) were detected in LNER cells with or without 4OHT treatment. - and ß-catenin were detected in LN cells and at similar levels with or without 4OHT in LNER cells, suggesting formation of a cadherin/catenin complex under either condition. p120ctn, which is stable in L cells lacking a cadherin, was present at similar levels in L, LN and LNER cells with or without 4OHT. Positions of molecular weight markers are indicated on the right. (C) Immunofluorescent light microscopic localization of NcadER in LNER cells with or without 4OHT. LNER monolayers plus or minus 100 nM 4OHT overnight were fixed and stained with anti-N-cadherin. The NcadER signal in cells with 4OHT appeared more organized and was particularly strong at sites of cell-cell contact, compared to cells without 4OHT. (D) Localization of N-cadherin in L cells and LN cells. As expected, L cells lacked N-cadherin, whereas LN cells exhibited strong N-cadherin staining, particularly at cell-cell borders. Bar, 50 µm.
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