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

FIGURE 8.

FIGURE 8.

Nedd4-2 facilitates internalization of NHE3. A, SK-CO15 cells were transfected with shCon, shNedd4-2, or HA-Nedd4-2. The steady-state expression levels of NHE3 were determined in the presence of cycloheximide. The graph shows quantification of NHE3 protein bands is shown. n ≥ 3. B, internalization of hNHE3, rbNHE3, and ratNHE3 was determined in PS120 fibroblasts. The graph shows the amounts of internalized NHE3 quantified as fractions of NHE3 in the surface membrane at the start of the experiment. *, p < 0.01. C, internalization of hNHE3 in PS120 cells transfected with shCon, shNedd4-2, or Nedd4-2 was determined. *, p < 0.01. NS, not significant. D, internalization of endogenous hNHE3 was determined in SK-CO15 cells, which were pretransfected with shCon, shNedd4-2, or Nedd4-2. *, p < 0.01 versus shNedd4-2. E, PS120 fibroblasts expressing hNHE3, rbNHE3, or ratNHE3 were treated with shNedd4-2 or shCon. Western blots show internalized NHE3 3 h after the temperature shift to 37 °C, which initiated internalization. Controls were kept at 4 °C for 3 h. The amount of internalized NHE3 was determined relative to internalized NHE3 in control cells. Representative Western blots of three independent experiments are shown.