Fig. 5. Only PLAP but not DPPIV is mislocalized in Caco2 cells after transient RNAi of TI-VAMP. Caco2 cells were electroporated with 60 pmol of siRNA targeting human TI-VAMP or against human β-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 human calreticulin (top panel, internal control for protein loading) and human TI-VAMP (bottom panel). TI-VAMP expression was decreased by more than 70% after transient RNAi of Caco2 cells on filters 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) Caco2 cells. (B) Cells were labeled with an antibody against PLAP and a secondary antibody coupled to FITC. Compared with its apical localization in Caco2 cells (left panel), PLAP is mislocalized to the lateral membrane in Caco2 TI-VAMP transient-knockdown cells (right panel). (C) Cells were labeled with antibodies against human DPPIV and secondary antibodies coupled to FITC. Note that, compared with FRT cells, the apical localization of DPPIV is unaffected by the transient knockdown of TI-VAMP, as is the basolateral localization of Ag525 (B, right panel). Bars, 10 µm.