Expression of S667F-kB3 in nonpolarized MDCK1 cells. (A-P) MDCK1 cells stably expressing normal kB3 or mutant S667F-kB3 that were grown on coverslips and fixed (A-L), or washed, incubated with the extracellular anti–band 3 antibody BRIC6 for 1 hour, and then fixed (M-P) as outlined in “Methods.” (A-L) Comparison of kB3 and S667F-kB3 localization with intracellular markers for the ER (calnexin) or TGN (TGN38). kB3 had only a partial overlap with the ER marker (merge; panel C) and some overlap with TGN38 (merge; panel I); most of the protein is at plasma membrane as previously reported.16 The majority of mutant S667F-kB3 immunoreactive protein overlapped with the calnexin (merge; panel F), but there was some overlap with the TGN38 staining (merge; panel L), suggesting that a small proportion of the protein reaches the late stages of the secretory pathway. (M-P) rbB3Ct staining (M,O) and BRIC6 staining (N,P). All cells expressing wild-type kB3 (confirmed by double staining with the rbB3Ct: panel M) are labeled with substantial amount of FITC-BRIC6 (N). Cells expressing S667F-kB3, which were detected with the rbB3Ct antibody (O), also bound a small amount of FITC-BRIC6 (P), suggesting that a small amount of S667F-kB3 can reach the plasma membrane. Scale bar equals 30 μm.
