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

FIGURE 6.

FIGURE 6.

Nedd4-2 binding is necessary for ubiquitination of hNHE3. A, the C-terminal truncation constructs of hNHE3 are shown. Numbers denote the amino acid position at the truncation site for each mutant. B, NHE3 variants were immunoprecipitated (IP, lower) followed by immunoblotting (IB) for HA-Nedd4-2 (upper). The relative amount of HA-Nedd4-2 co-immunoprecipitated was quantified with the interaction with full-length hNHE3-834 as 100%. C, cells expressing hNHE3 variants were transfected with HA-Ub, and NHE3 was immunoprecipitated. Ubiquitination levels are shown. D, surface expression of hNHE3 variants was determined by surface biotinylation. Cells were transfected with shNedd4-2 (+) or shCon (−) before surface biotinylation. Data are representative of three independent experiments. Quantification was done with the control transfected (−) lane of the respective samples as 100%. E, the effects of Nedd4-2 knockdown on Na+-dependent pHi recovery of hNHE3-729 and hNHE3-710 were determined. n = 9. *, p < 0.01 compared with shCon.