Fig. 4. The RRR ER-retention/retrieval signal reduces surface expression of Na_v1.8. (A,B) COS-7 cells transfected with a plasmid expressing Myc-CD8, Myc-CD8-KKTN, or with a series of constructs with the C-terminus of CD8 linked to 2L or to the various segments of 1L of Na_v1.8, were subjected to cell-surface biotinylation/immunoblotting. Representative immunoblot and quantitative analyses are shown. Actin served as an internal control for protein loading. The ratio of immunoblot intensity of surface versus total protein was calculated and data were plotted as a percentage of the controls (n3). ^**P<0.01 versus cells transfected with the plasmids indicated; ^##P<0.01 versus Myc-CD8-1L-1-1-expressing cells. (C) HEK293 cells were transfected with plasmids expressing Na_v1.8 or Na_v1.8 mutants in which 495RRR497 was mutated to alanine (Na_v1.8m-Myc), or the first intracellular loop was replaced by the second one (Na_v1.8r-Myc), and subjected to cell-surface biotinylation/immunoblotting. Data were plotted as a percentage of Na_v1.8-Myc. ^*P<0.05 versus cells transfected with Na_v1.8-Myc (n=3).