Fig. 6. Insolubilization of occludin after Ca²⁺ switch in MDCK cell lines expressing L-ASIP WT (A,C) or PB (B,D). MDCK cells cultured with or without 10 ng/ml of DC were harvested at different times after Ca²⁺ switch, and the NP-40-insoluble fractions were subjected to western blot analysis with anti-ASIP (C2-3AP) or anti-occludin pAb. CBB staining of tubulins on the blotted membranes is shown as a control. The results are representative of three independent experiments. NP-40-insoluble occludin was detected by chemiluminescence ECL, and the resulting signals were quantified directly with LAS-1000 plus system (FUJI Photo Film, Tokyo, Japan) in a fluorescence image mode and normalized to the amount of tubulins in each lane (C,D). Each point is the mean±s.e.m. of three independent experiments. P-values were calculated with a two-sided t-test and statistical significance was considered at P<0.05 (asterisk). Induced overexpression of L-ASIP WT (C; filled circles), but not PB (D; filled squares), results in the rapid accumulation of occludin in NP-40-insoluble fractions after Ca²⁺ switch.