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

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© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Nedd4-2 regulates ubiquitination of hNHE3. A, PS120 cells fibroblasts expressing human, rabbit, opossum, or rat NHE3 were transiently transfected with HA-Ub. NHE3 was immunoprecipitated (IP) using VSVG antibody, and the levels of NHE3 ubiquitination were determined by immunoblotting (IB) with a HA-Ub antibody. B, PS120/NHE3 cells were transfected with shCon or shNedd4-2. NHE3 was immunoprecipitated (lower panel) with anti-VSVG antibody followed by immunoblot using P4D1 to detect NHE3 ubiquitination (upper panel). Data are representative of at least three experiments. C, PS120/NHE3 cells were transiently transfected with HA-Ub and lentiviral shNedd4-2. shCon was used as a control. Ubiquitination of immunoprecipitated NHE3 was determined using anti-HA antibody. The bottom panel shows the total amounts of immunoprecipitated NHE3. D, ubiquitination of hNHE3 in SK-CO15 cells transfected with shCon or shNedd4-2 was determined.