Figure 4.
Calmodulin does not regulate FcRn polarity. (A) MDCK cell clones expressing WT or mutant FcRn were cultured on Transwell filters, and FcRn was detected with an anti-HA mouse mAb (green), ZO-1 with a rabbit anti-ZO-1 polyclonal antibody (blue), and F-actin with phalloidin-Texas Red (red). Bar, 100 μm. A control clone carrying empty expression plasmids (vector control) was also stained. X-Z sections are depicted. (B) MDCK cell clones expressing WT or mutant FcRn were cultured on Transwell filters and the surface expression of FcRn was examined by selective cell surface biotinylation. Biotinylated FcRn (Memb; lanes 1, 2, 5, and 6) and cellular FcRn (WCL representing 1% of the total whole cell lysate; lanes 3, 4, 7, and 8) were detected by immunoblot. Blots were reprobed for GP135. Two clones per construct were examined, and data are representative of four independent experiments. (B) Band intensities were quantified by densitometry, and the percentage of apical or basolateral FcRn relative to total cellular FcRn present in whole cell lysates is expressed as mean ± SEM. Data from two clones per construct were combined. NS, nonstatistically different (p > 0.05).