Fig. 2. DPPIV but not Ag 35/40 kDa is mislocalized in fully polarized stable FRT clones lacking TI-VAMP. FRT cells stably expressing PLAP (FRT PLAP), and FRT cells stably expressing PLAP and a vector where the siRNA targeting rat TI-VAMP was introduced (FRT si TI-VAMP::PLAP), were grown on filters for 4 days. (A) Cell extracts (40 µg) from FRT PLAP and FRT si TI-VAMP::PLAP were analyzed by SDS-PAGE and western blotting with antibodies against PLAP (lanes two and four) and human TI-VAMP (lanes one and three). Note that the two FRT cells express similar amounts of PLAP, whereas TI-VAMP is constitutively knocked-down in FRT si TI-VAMP::PLAP compared with FRT PLAP. (B) Confocal Z and X-Y sections (top and bottom) of FRT PLAP and FRT si TI-VAMP::PLAP cells were labeled with antibodies against rat DPPIV (left panel) and Ag 35/40 kDa (right panel) and secondary antibodies coupled to FITC. Note that DPPIV is only mislocalized in FRT si TI-VAMP::PLAP cells, whereas the basolateral localization of Ag 35/40 kDa is unchanged in both cell lines. Bars, 10 µm.