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. 2012 Jul 30;287(39):32940–32952. doi: 10.1074/jbc.M112.353334

FIGURE 3.

FIGURE 3.

The galectin lattice increases the N-cadherin-GFP mobile fraction at cell-cell junctions. A, Mgat5+/+ or Mgat5−/− cells were transfected with N-cadherin-GFP (Nc-GFP) and treated or not with lactose (+Lac) for 48 h before FRAP analysis (bleached zone is circled). Boxed regions are shown before bleach, immediately after bleach (0 s), and 120 s after bleach. Bar: 20 μm. B, FRAP analysis of N-cadherin-GFP was performed on Mgat5+/+, Mgat5−/−, or Mgat5−/−Res either untreated (UT) or following 48 h of sucrose (+Suc), lactose (+Lac), or swainsonine (+SW) treatment. 12–15 points were acquired from 10–15 cells, and the mean percentages of recovery of N-cadherin-GFP in individual cell-cell junctions are presented as a fitted curve according to the equation Y = Ymax × (1 − exp(−0.69/X × t½)); one of three independent experiments is represented here. A significant decrease in the plateau of the percentage of recovery is observed after lactose or swainsonine treatment in comparison with the sucrose or untreated cells (maximum recovery percentage values (Ymax)) of untreated or sucrose-treated when compared with lactose or swainsonine treatment (p < 0.05). C, FRAP analysis of N-cadherin-GFP in Mgat5+/+, Mgat5−/−, and Mgat5−/−Res cells 48 h after transfection with control siRNA (siCtl) or siRNA against Gal-3 (siGal-3). Untreated cells or siCtl cells displayed a significant higher Ymax values in comparison with the siGal-3-treated cells. D, FRAP analysis of N-cadherin-GFP in Mgat5+/+ cells transfected with control (siCtl) or Gal-3 targeting (siGal-3) siRNA. After 48 h, cells were incubated 10 min with Gal-3 before performing FRAP. Statistical analyzes revealed that the addition of Gal-3 did not affect the Ymax value of siCtl-treated cells but rescued the effect of siGal-3 treatment. Means of percentages of recovery of 12–15 individual cell-cell junctions are presented as fitted curves. Experiments were reproduced independently at least three times. See supplemental Table S1 for analysis of FRAP data from all experiments. Significant p values from Student's t test are indicated. Error bars indicate ± S.E.