Fig. 4. Characterization of MDCK Tet-Off cell lines expressing T7-tagged L-ASIP WT or PB. (A) Schematic structures of the constructs. The aPKC-binding sequence is found in L-ASIP WT but not in L-ASIP PB. (B) Each cell line was cultured with (+) or without (-) 1.0 µg/ml of tetracycline (TC), and 5x10⁴ cells/lane were subjected to western blot analysis with the T7-tag-specific antibody (Omni probe) or anti-aPKC antibody. The expression of T7-tagged ASIP/PAR-3 in each cell line was strongly induced by the withdrawal of TC, whereas 1.0 µg/ml of TC completely repressed the expression to under the detectable levels. (C-E) Immunofluorescent images show that the overexpressed T7-tagged ASIPs localize mainly at cell-cell contacts of MDCK cells cultured without TC (TC0). Confluent monolayers of MDCK cells expressing T7-L-ASIP WT (C) or T7-L-ASIP PB (D) were labeled by the Omni probe pAb. 1 µg/ml of TC (TC1) completely repressed the expressions of L-ASIP WT (E) and L-ASIP PB (not shown). Bars, 100 µm; 10 µm (inset).