FIGURE 9.
Effect of CHP3 on abundance of the cell surface fully glycosylated form of NHE1. AP-1 cells were transfected with a fixed amount of expression plasmid DNA containing NHE1HA and an increasing ratio of CHP3myc to empty expression vector (pCMV). Cells were also cotransfected with a plasmid that constitutively expresses green fluorescent protein (pGFP) as a control for transfection efficiency. At 36 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 NHE1HA and CHP3myc were detected as described in Fig. 6. GFP was detected using a primary rabbit polyclonal anti-GFP antibody 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.