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Fig. 1. Apical but not basolateral proteins are mislocalized in fully polarized FRT cells after transient RNAi of TI-VAMP. FRT cells stably expressing PLAP were electroporated with 100 pmol of siRNA targeting rat TI-VAMP or against human 
-globin and grown on filters for 4 days. (A) Cell extracts (40 µg) from control (siRNA beta glob) and TI-VAMP transient-knockdown (siRNA TI-VAMP) cells were analyzed by SDS-PAGE and western blotting with antibodies against calreticulin (top panel, internal control for protein loading) and human TI-VAMP (bottom panel). The decrease in TI-VAMP expression in transient-knockdown cells was calculated to be 
88% in three different experiments. (B,C) Confocal Z and X-Y sections (top and bottom) of control (siRNA beta glob) and TI-VAMP transient-knockdown (siRNA TI-VAMP) cells. (B) Cells were labeled with antibodies against rat DPPIV (left panel) and Ag 35/40 kDa (right panel), and secondary antibodies were coupled to FITC. Note that the apical localization of DPPIV in controls switches in TI-VAMP transient-knockdown cells to the lateral membrane (Z section and X-Y section bottom panel), whereas the basolateral localization of Ag 35/40 kDa is unchanged. (C) Cells were labeled with antibodies against PLAP and secondary antibodies coupled to FITC. Note that, as for DPPIV, PLAP is mislocalized to the lateral membrane. Bars, 10 µm.
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