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).
