Figure 6.

Limited proteolysis assay showing the protective effect of 14-3-3 to pNedd4-2^186−975. (A) Phosphorylated Nedd4-2^186−975 in the absence and presence of 14-3-3η digested with trypsin for 10, 20, and 30 min. The protease/Nedd4-2 ratio was 1:1000 (w/w). The reactions were stopped by boiling the samples with SDS/PAGE loading buffer at the times indicated before SDS-PAGE analysis. The black rectangle marks the quantified region. Unedited gel image is provided in supporting material (Fig. S15). (B) The density of the bands representing degraded Nedd4-2^186−975 in the presence and absence of 14-3-3η were quantified in ImageLab (Bio-Rad). Error bars represent the standard deviation of four independent experiments. Asterisks indicate significant differences, according to Student's t-tests comparing relative changes between samples with and without 14-3-3η at selected time points (^∗p ≤ 0.01, ^∗∗∗p ≤ 0.0001).