FIGURE 6.

CHP3-mediated trafficking of NHE1 to the cell surface is not dependent on N-myristoylation or Ca²⁺ binding of CHP3. AP-1 cells were grown to subconfluence on 10-cm dishes and then transiently transfected with a fixed amount of expression plasmid DNA containing NHE1_HA (8 μg) and an increasing ratio of CHP3_myc (WT, G2A, or D123A) to empty expression vector (pCMV) (0–2 μg per dish). Cells were also simultaneously cotransfected with a plasmid that expresses green fluorescent protein (pGFP) as a control for transfection efficiency. At 48 h post-transfection, cells were subjected to surface biotinylation as described under “Experimental Procedures,” and whole cell lysates were prepared. A major fraction of the lysates was subsequently incubated with NeutrAvidin-Sepharose beads to isolate the biotinylated cell surface proteins. Aliquots of the whole cell lysates and biotinylated cell surface proteins were subjected to SDS-PAGE and immunoblotting. Expression of fully glycosylated (fg) and core-glycosylated (cg) forms of NHE1_HA and CHP3_myc were detected as described in Fig. 4. GFP was detected using a primary rabbit polyclonal anti-GFP antibody (α-GFP) and a secondary goat anti-rabbit antibody conjugated to horseradish peroxidase. Immunoblots of the lysates were stripped and reprobed for endogenous GAPDH as a control for protein loading. Data shown are representative of three independent experiments.