(a) Cytotrap yeast two hybrid (Y2H) indicating an interaction between Cops3 and the cytoplasmic tail of Dsg1. Growth at 25°C is permissive; growth on galactose (Gal) at 37°C confirms interaction. (b) Cops4 of the CSN does not interact with Dsg1. A known binding partner of Dsg1, Plakoglobin (Pg), was used as a positive control. (c) Purified GST-Dsg1 (includes the entire cytoplasmic domain), was bound to sepharose beads and incubated with normal human keratinocyte (NHEK) lysates. (d) An endogenous Dp immunoprecipitation (IP) was performed in NHEKs after 1 day of differentiation, indicating an interaction with Cops3. (e) A 32 kDa S-tagged C-terminal truncation of Dp (DP-S-tag) associates with Cops3 in squamous cell carcinoma line 9 (SCC9) lysates. (f) Structured Illumination Microscopy (SIM) of Cops3 (red) and Dsg1 or Dp (green) in NHEKs after 2 days of differentiation, shows close proximity between Cops3 and desmosome components at junctions. (g and h) Proximity ligation assay (PLA) in NHEK cultures with siRNA-targeting COPS3, DSG1, DP, or scramble sequence (siCONT) after 1 day of differentiation. Quantification is displayed to the right of the image panels. Cops3/Dsg1= *p<0.001, and Cops3/Dp= *p<0.00005, Student’s t test; mean ±SEM. (i) PLA in NHEK cultures after 1 day of differentiation with adherens junction components (Ecadherin, E-cad, Beta catenin, B-cat, Alpha catenin, A-cat) and co-stained with Plakoglobin to mark cell borders. Proximal proteins within 40–100 nm appear as red but have been pseudo-colored yellow in (g) and (h) and have been dilated in (i) for ease of visualization in Fiji. All experiments are representative of 3 or more independent repeats. Scale bars = 20 μm.