Fig. 9. Effects of LNX1p80 expression on the amount of endogenous claudin-1 protein in MDCK cells. (A) Reduction in the amount of claudin-1 by LNX1p80 expression in MDCK cells. Western blot of the whole lysate (whole), 1% NP-40-soluble (sup) and insoluble (pellet) fraction of MDCK cells constitutively expressing EGFP or EGFP-LNX1p80 with anti-claudin-1 pAb. The amount of NP-40-insoluble claudin-1 (pellet) was remarkably reduced in cells expressing EGFP-LNX1p80 compared with that in EGFP-expressing MDCK cells. (B) The effect of RING-domain mutant of LNX1p80 on claudin-1. Tet-EGFP-LNX1p80 MDCK cells and Tet-EGFP-LNX1p80C48A MDCK cells were cultured with (+) or without (–) doxycycline. The amounts of claudin-1, E-cadherin, EGFP-LNX1p80 and EGFP-LNX1p80C48A in total cell lysates were analyzed by immunoblotting with anti-claudin-1 pAb, anti-E-cadherin mAb and anti-GFP mAb. The total claudin-1 protein level was decreased in EGFP-LNX1p80-expressing cells, but not in EGFP-LNX1p80C48A-expressing cells. The amount of E-cadherin did not change in this experiment. (C) Immunofluorescence localization of claudin-1 in MDCK cells expressing EGFP-LNX1p80C48A. Tet-EGFP-LNX1p80C48A MDCK cells cultured without doxycycline were immunostained with anti-claudin-1 pAb. Vertical sections were obtained by confocal microscopy (lower panel). The concentration of claudin-1 was not limited to TJ regions but also extended to the lateral membrane (arrow). EGFP-LNX1p80C48A was colocalized with claudin-1. Scale bar: 10 µm.