FIGURE 9:

NHE3-S719A predominantly associates with smaller protein complexes. Total cell lysate was prepared from Caco-2/bbe cells grown on Transwell filters and infected with Ad-HA-NHE3-WT, NHE3-S719A, or NHE3-S719D. The ∼2-mg total cell lysates were subjected to complex size analysis using sucrose gradient centrifugation as described in Materials and Methods. Because samples were collected from the bottom, starting complex sizes are larger. NHE3 was probed with HA mouse mAb. Distribution of NHE3-WT and S719D by size in protein complexes is similar, but S719A is predominantly in smaller protein complexes. A representative experiment, which was repeated three times with similar results, is shown.