Fig. 7. DPPIV but not PLAP is mislocalized in Caco2 cells after transient RNAi of VAMP8. Caco2 cells were electroporated with 60 pmol of siRNA targeting human VAMP8 or against human -globin and grown on filters for 4 days. (A) Cell extracts (40 µg) from control (siRNA beta glob) and VAMP8 transient-knockdown (siRNA VAMP8) cells were analyzed by SDS-PAGE and western blotting with antibodies against human calreticulin (top panel, internal control for protein loading) and human VAMP8 (bottom panel). VAMP8 expression decreased by more than 80% 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 VAMP8 transient-knockdown (siRNA VAMP8) 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 unaffected by the transient knockdown of VAMP8. (C) Cells were labeled with antibodies against human DPPIV (left panel) and secondary antibodies coupled to FITC. Note that, compared with FRT cells, the apical DPPIV is also mislocalized to the lateral membrane in Caco2 VAMP8 transient-knockdown cells (right panel), whereas the basolateral localization of Ag525 is unaffected (B, right panel). Bars, 10 µm.