Dsc2 associates with kinesin-2. (A) Dual-label immunofluorescence confocal microscopy showing Dsc2 (right) and Dsg2 (left) with KIF3A. A subset of Dsc2-positive particles colocalize with KIF3A (yellow circles), whereas Dsg2-positive particles do not colocalize with KIF3A (blue circles). Boxes showed the zoomed areas. Bars, 10 µm. (B) Recombinant Dsg2 cytoplasmic tail–GST or Dsc2 cytoplasmic tail–GST were incubated with Scc9 cell lysates, and retained proteins were immunoblotted for the presence of KIF3A. KIF3A associates with Dsc2 tail–GST but not Dsg2 tail–GST. Note that the blot is the same as on Fig. 2 C. (C) PLA was performed using primary antibodies directed against Dsg2 or Dsc2 coupled with KIF3A antibodies on nontargeting (NT) siRNA (siNT; top) or siKIF3A-treated Scc9 cells (middle). Red particles were quantified, showing significant Dsc2–KIF3A interactions (***, P < 0.001) compared with Dsg2–KIF3A, which were abrogated upon KIF3A knockdown (bottom). Number of cells per condition = ∼240. Bars, 20 µm. (D) Level of KIF3A knockdown (KD) in the PLA experiment shown above, representative of three experiments on the right (**, P < 0.01). Protein expression level of Dsg2, Dsc2, and E-cadherin (E-cad) shown in the bottom middle were unchanged. Error bars represent SEM. Tub, tubulin.