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. 2014 May 15;289(26):18360–18372. doi: 10.1074/jbc.M113.541706

FIGURE 5.

FIGURE 5.

Nedd4-2 regulates hNHE3 surface membrane expression. A, surface biotinylation of PS120/NHE3 cells transfected with shCon (−) or shNedd4-2 (+) was performed as described under “Experimental Procedures.” NHE3 expression in the surface membrane (S-NHE3) and total lysate (T-NHE3) was determined by immunoblotting using VSVG antibody. β-Actin expression in lysates was used as a loading control. The expression levels of S-NHE3 were quantified relative to each species transfected with shCon. B, surface expression of NHE3 was determined in SK-CO15 cells transfected with shCon or shNedd4-2. EM450 was used for immunoprecipitation and subsequent immunoblotting for NHE3. C, ubiquitination of NHE3 in PS120/hNHE3 and PS120/rbNHE3 cells was determined by isolation of surface NHE3 by biotinylation followed by immunoblotting (IB) for Ub using P4D1. IP, immunoprecipitation. D, in PS120/hNHE3 expressing HA-Nedd4-1 or HA-Nedd4-2, surface membrane was separated by surface biotinylation. NHE3 was immunoprecipitated using anti-VSVG antibody from the surface fraction followed by immunoblotting with anti-HA antibody. Immunoprecipitated Nedd4-2 is indicated by an arrow. In all lanes a nonspecific band slightly smaller than HA-Nedd4-1 was visible. The bottom panels show the presence of HA-Nedd4-1, HA-Nedd4-2, and NHE3 in the surface fraction. Representative figures from three independent experiments are shown.