Figure 7. ARVC mutation disrupts the Dsg2 tail–tail interaction and leads to rapid internalization. (A) ARVC mutations were introduced into pSOS-DUR vector individually. The interaction between the myristoylation-DUR and the SOS-DUR WT or SOS-DUR with mutation was tested in the CytoTrap two-hybrid system. Representative results from one experiment are shown. (B) HL-1 cells were transiently transfected with Dsg2.FL-WT or Dsg2.FL-V997fsX1016 with Lipofectamine. 48 h after transfection, cell lysates were collected and immunoblotted with the indicated antibodies. The 6D8 antibody exclusively recognizes the ectopic human Dsg2 constructs. (C) HL-1 cells expressing Dsg2.FL-WT or Dsg2.FL-V997fsX1016 were stained with the 6D8 antibody. Bar, 20 µm. (D) Mutant-expressing cells were labeled with the 6D8 antibody at 4°C (asterisks) and then incubated at 37°C for 0 or 30 min. Residual surface antibodies were subsequently stripped. Internalized molecules (arrows) were visualized using indirect immunofluorescence. Bar, 20 µm. Graph shows the ratio of mean cytoplasmic intensity per cell/mean surface label intensity per cell for each mutant. Error bars are SEM. n = 3. Paired t test. Gal, galactose; Glu, glucose.
Figure 7.

ARVC mutation disrupts the Dsg2 tail–tail interaction and leads to rapid internalization. (A) ARVC mutations were introduced into pSOS-DUR vector individually. The interaction between the myristoylation-DUR and the SOS-DUR WT or SOS-DUR with mutation was tested in the CytoTrap two-hybrid system. Representative results from one experiment are shown. (B) HL-1 cells were transiently transfected with Dsg2.FL-WT or Dsg2.FL-V997fsX1016 with Lipofectamine. 48 h after transfection, cell lysates were collected and immunoblotted with the indicated antibodies. The 6D8 antibody exclusively recognizes the ectopic human Dsg2 constructs. (C) HL-1 cells expressing Dsg2.FL-WT or Dsg2.FL-V997fsX1016 were stained with the 6D8 antibody. Bar, 20 µm. (D) Mutant-expressing cells were labeled with the 6D8 antibody at 4°C (asterisks) and then incubated at 37°C for 0 or 30 min. Residual surface antibodies were subsequently stripped. Internalized molecules (arrows) were visualized using indirect immunofluorescence. Bar, 20 µm. Graph shows the ratio of mean cytoplasmic intensity per cell/mean surface label intensity per cell for each mutant. Error bars are SEM. n = 3. Paired t test. Gal, galactose; Glu, glucose.

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