Figure 5

Loop and Cter of CALHM1 and 3 contain basolateral sorting signals. (A) Illustration of chimeric constructs of the intracellular domain-truncated invariant chain (IiΔN) or CD4ΔC and an intracellular domain of mouse CALHM1 or 3. All chimeras were FLAG-tagged on their Cter. (B) Effects of MG132 (20 µg/mL) on expression levels of the chimeric proteins. Upper panels display representative Western blots of CD4ΔC-CALHM1Cter and CD4ΔC-CALHM3Cter, and the lower panel shows a quantitative summary of expression levels of each chimera 0, 4, and 8 h after MG132 treatment. N = 3. Uncropped blots are shown in Supplementary Fig. S2. (C) Representative Western blots of the domain-specific biotinylation assays in stable MDCKII clones expressing each chimera. Whole-cell lysates (Input) of apically (Ap)- or basolaterally (Bl)-biotinylated cells and their avidin pull-down eluates (Surface) were analyzed by SDS-PAGE/Western blotting. FLAG-tagged chimeric proteins were detected by anti-FLAG antibody. Na⁺/K⁺ ATPase and β-tubulin were detected as markers of the basolateral membrane and cytosol, respectively. IiΔN and CD4ΔC alone were tested as controls. CD4ΔC-CALHM1Cter and CD4ΔC-CALHM3Cter were analyzed after 4-h MG132 treatment (+MG). Images taken from the same gel but at different exposures are separated by dashed black lines. Uncropped blots are shown in Supplementary Fig. S3. (D) 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 = 4~5. ^NSp > 0.05, *p < 0.05 and **p < 0.01 (vs. control, Bonferroni post-hoc test).