DUR inhibits Dsg2 internalization. (A) Lysates from mutant-expressing SCC68 cells were immunoblotted with the indicated antibodies. The epitope for 6D8 is in the Dsg2 ectodomain. (B) Biotinylation assay was performed in mutant-expressing SCC68 cells grown in medium with 0.25 mM Ca²⁺. L, surface pool of the Dsg2 mutants. 30’, cytoplasmic pool of the Dsg2 mutants after 30 min of internalization. S, residual Dsg2 mutant protein left on the cell surface after stripping. Asterisks indicate FL proteins, and circles show cleavage products. Densitometric analysis was performed using ImageJ to determine the ratio of cytoplasmic/surface intensity. Numbers at the bottom indicate the internalization ratio for each mutant. (C) Collective results from seven independent biotinylation assays performed at 0.25 mM Ca²⁺. n ≥ 4 per mutant. Paired t test. The bottom and top of the box are the 25th and 75th percentile (the lower and upper quartiles), respectively. The horizontal line near the middle of the box is the 50th percentile (the median). The ends of the whisker are the minimum and maximum of all the data. (D) Biotinylation assay of SCC68 cells expressing Dsg2.ICS or Dsg2.RUDI grown in medium with 0.25 mM Ca²⁺ with varying internalization times: 2, 10, and 20 min. (E) SCC68 cells expressing Dsg2.ICS or Dsg2.RUDI were pretreated with 10 µg/ml cycloheximide for 30 min (time 0) and then kept in cycloheximide-containing medium for an additional 4 or 8 h before collection. Cell lysates were immunoblotted for FLAG. (F) SCC68 cells expressing Dsg2.ICS or Dsg2.RUDI were grown in medium containing 0.09, 0.25, or 1.0 mM Ca²⁺ and subjected to biotinylation assay with 30-min internalization. (G) Biotinylation assay conducted on SCC9 cells expressing the Dsg2 mutants.