Fig. 3.
Occludin interacts with Nedd4–2 via its conserved NH2-terminal PY motif. A and B: IP in HEK293 cells transfected with Flag-tagged occludin or occludin Mut with and without Xpress-tagged Nedd4–2. Protein lysates were subject to IP using an ANTI-FLAG Affinity Gel and then analyzed by immunoblotting using anti-Xpress and anti-Flag. Nedd4–2 coprecipitates with occludin and a PY mutation (PPPY9–12 to PAAY) significantly reduces coprecipitated Nedd4–2 proteins. Representative blot in A and pooled densitometric data from 3 experiments in B. Immunoprecipitated Nedd4–2 proteins were first expressed as a ratio of the immunoblottable Nedd4–2 and then normalized to the amount of immunoprecipitated occludin or OccMut in lysates used for the IP reaction and then compared with wild-type occludin that was set at 100%. Means ± SE, **P < 0.001, Tukey's pairwise multiple comparison.