Identification of functional basolateral sorting signals. (A) Representative Western blots of the domain-specific biotinylation assays in stable clones of MDCKII cells expressing CD4ΔC, CD4ΔC-CALHM1Loop, CD4ΔC-CALHM1Cter, or either one of the mutant chimeras in which one of the candidate basolateral sorting signals was disrupted by alanine substitution. Whole-cell lysates (Input) of apically (Ap)- or basolaterally (Bl)-biotinylated cells and their avidin pull-down eluates (Surface) were analyzed by Western blotting. CD4ΔC fusion proteins were detected by anti-FLAG antibody. Na+/K+ ATPase and β-tubulin signals serve as markers for the basolateral membrane and cytosol, respectively. CD4ΔC-CALHM1Cter proteins were analyzed after 4-h MG132 treatment (+MG). Uncropped blots are shown in Supplementary Fig. S4. (B) The amounts of each chimera detected in the apical and basolateral membranes are expressed as percentages of the total amount in the entire plasma membrane. N = 4~7. NSp > 0.05 **p < 0.01 and ***p < 0.001 (Bonferroni post-hoc test).