Dsg2 associates with kinesin-1. (A) Dual-label confocal microscopy showing Dsg2 (left) and Dsc2 (right) with KHC. Under these experimental conditions, the KHC antibody results in a combination of particulate and fibrous staining, the latter likely representing MTs. A subset of Dsg2-positive particles colocalize with KHC (yellow circles), whereas Dsc2-positive particles do not colocalize with KHC (blue circles). Boxes showed the zoomed areas. Bar, 10 µm. (B) Myc-tagged KIF5B was transfected into Scc9 cells, subjected to immunoprecipitation, and immunoblotted for Dsg2 and Dsc2. Dsg2, but not Dsc2, associated with KHC-Myc. (C) Recombinant Dsg2 cytoplasmic tail–GST or Dsc2 cytoplasmic tail–GST were incubated with Scc9 cell lysates. Retained proteins were immunoblotted for the presence of KHC. KHC associates with Dsg2 tail–GST but not Dsc2 tail–GST. (D) Recombinant His-tagged KHC tail was incubated with Scc9 cell lysates, and retained proteins were tested for the presence of desmosomal cadherins. Dsg2, but not Dsc2, associates with the KHC tail. (E) Proximity ligation assay (PLA) was performed as described in Materials and methods (bottom left) using primary antibodies directed against Dsg2 or Dsc2 coupled with KHC antibodies on nontargeting (NT) siRNA (siNT; top) or siKHC-treated Scc9 cells (middle). Red-labeled particles indicating protein complex formation were quantified (bottom right) to show significant Dsg2–KHC interactions compared with Dsc2–KHC (number of cells per condition = ∼195; ***, P < 0.001), which were abrogated upon KHC knockdown. Bars, 20 µm. (F) Level of KHC knockdown (KD) in PLA experiment shown above, representative of five experiments quantified below (**, P < 0.01). Protein expression levels of Dsg2, Dsc2, and E-cadherin (E-cad) shown in the top right do not change. Error bars represent SEM. Ab, antibody; Tub, tubulin.