Knockdown of KIF3A or KAP3 affects Dsc2, but not Dsg2, accumulation at junctions in Scc9 cells. KIF3A and KAP3 (kinesin-2 intermediate chain) functions were targeted with shRNA or siRNA, respectively. (A) Immunoblot demonstrating ∼50% reduction of KIF3A in shRNA-transfected cells (n = 3; *, P < 0.05). α-Tubulin (Tub) is the loading control. (B) Immunoblot demonstrating ∼60% decrease in KAP3 in siRNA-transfected cells (n = 5; *, P < 0.05). No change in expression of Dsg2 and Dsc2 was observed. (C) Scc9 cells were transiently transfected with a GFP-shRNA construct against KIF3A or nontargeting (NT) shRNA for 2 d, replated and, after 24 h, fixed and labeled either for Dsg2, Dsc2, or E-cadherin (E-cad). Knockdown (KD) of KIF3A (cells with GFP) delays Dsc2 recruitment into newly forming junctions but does not have a measurable effect on Dsg2 or E-cadherin localization at cell–cell borders. (D) Line scan analysis of border intensities for Dsc2, Dsg2, and E-cadherin were performed for ∼50 borders per each condition with three times scans per border (n = ∼150 for each condition). Borders were chosen randomly from three independent experiments per condition. KIF3A knockdown results in a decrease of peak intensity for Dsc2 at cell–cell borders compared with the control cells (P = 0.01). (E) Scc9 cells were transfected with siRNA oligos against KAP3 or control siRNA for 2 d, replated, and after 24 h, labeled either for Dsg2 or Dsc2. (F) KAP3 knockdown results in loss of the peak intensity for Dsc2 at the cell–cell border (P < 0.001). pxl, pixel. Bars, 20 µm.