Figure 5. DUR controls Dsg2 complex... | Rockefeller University Press

$Figure 5. DUR controls Dsg2 complex profile and mediates Dsg2 tail–tail interactions. (A) Cells were crossed-linked with 20 µg/ml dithiobis(succinimidylpropionate) at 4°C for 10 min, lysed in 0.5× USB, and then subjected to sucrose gradient fractionation (13 to 4% discontinuous sucrose gradient in 0.5× USB; 150,000 g for 18 h at 20°C). Fractions were collected starting from the bottom of the tube and blotted with antibodies against IL2R (top) or FLAG (middle and bottom). Signal intensity of each fraction was measured by densitometric analysis. To calculate the percentage of total loading per fraction, the intensity of a fraction is divided by the total intensity of all the fractions. The data shown are from one representative experiment out of two independent repeats. (B, a) In situ PLA was conducted on Dsg2-ICS– or Dsg2-RUDI–expressing cells using 4B2 and anti-FLAG antibodies. Bar, 60 µm. (b) The integrated signal intensity per image was measured and plotted. Graph was from one representative experiment with number of images quantified: Dsg2-ICS (n = 77) and Dsg2-RUDI (n = 78). The bottom and top of the box are the 25th and 75th percentile (the lower and upper quartiles), respectively. The horizontal line near the middle of the box is the 50th percentile (the median). The ends of the whisker are the minimum and maximum of all the data. (C, top) Schematic representation of Dsg2 fragments. (bottom) Analysis of the Dsg2 fragments using a yeast CytoTrap two-hybrid system. The data shown here were from one representative experiment out of three independent repeats. Gal, galactose; Glu, glucose; TD, terminal domain; L, linker domain.$

Figure 5.

DUR controls Dsg2 complex profile and mediates Dsg2 tail–tail interactions. (A) Cells were crossed-linked with 20 µg/ml dithiobis(succinimidylpropionate) at 4°C for 10 min, lysed in 0.5× USB, and then subjected to sucrose gradient fractionation (13 to 4% discontinuous sucrose gradient in 0.5× USB; 150,000 g for 18 h at 20°C). Fractions were collected starting from the bottom of the tube and blotted with antibodies against IL2R (top) or FLAG (middle and bottom). Signal intensity of each fraction was measured by densitometric analysis. To calculate the percentage of total loading per fraction, the intensity of a fraction is divided by the total intensity of all the fractions. The data shown are from one representative experiment out of two independent repeats. (B, a) In situ PLA was conducted on Dsg2-ICS– or Dsg2-RUDI–expressing cells using 4B2 and anti-FLAG antibodies. Bar, 60 µm. (b) The integrated signal intensity per image was measured and plotted. Graph was from one representative experiment with number of images quantified: Dsg2-ICS (n = 77) and Dsg2-RUDI (n = 78). 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. (C, top) Schematic representation of Dsg2 fragments. (bottom) Analysis of the Dsg2 fragments using a yeast CytoTrap two-hybrid system. The data shown here were from one representative experiment out of three independent repeats. Gal, galactose; Glu, glucose; TD, terminal domain; L, linker domain.

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