Figure 1.

Expression of Na,K-ATPase β-subunit in MSV-MDCK cells. (A-D) Expression levels of Na,K-ATPase α₁- and β₁-subunit. Total cell lysates of MSV-pCDNA3, MSV-NaKβ-cl 1, and MSV-NaKβ-cl 2 cells were separated on a 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-Na,K-ATPase β-subunit (A) or α-subunit (C) antibody. The blots from two gels each for β-subunit (B) and α-subunit (D) were quantified by densitometric analysis. The α- and β-subunit expression levels in MSV-NaKβ cells were compared with the α- and β-subunit levels in MSV-pCDNA3 cells. Bars, means ± SE. (E-K) Cell surface expression of Na,K-ATPase α- and β-subunit. (E-G) Immunofluorescence of Na,K-ATPase α-subunit. Confluent monolayers of MSV-pCDNA3 (E), MSV-NaKβ-cl 1 (F), and MSV-NaKβ-cl 2 (G) cells were washed, fixed, and stained with anti-Na,K-ATPase α-subunit and FITC-conjugated anti-mouse secondary antibody. Bar, 20 μm. (H-K) Cell surface biotinylation. Cell surface proteins of MSV-pCDNA3, MSV-NaKβ-cl 1, and MSV-NaKβ-cl 2 cells were labeled with membrane-impermeable Sulfo-NHS-biotin and biotinylated proteins were precipitated with streptavidin-conjugated beads. The precipitates were separated by SDS-PAGE and immunoblotted for Na,K-ATPase α-subunit (J) or β-subunit (H). The quantitative data of α-subunit (K) and β-subunit (I) cell surface expression of two independent experiments are shown. Bars, means ± SE. (L-M) Na,K-ATPase function in β-subunit expressing MSV-MDCK cells. Ouabain-sensitive ⁸⁶Rb⁺ flux measurements (L) and atomic emission spectrometry to determine intracellular Na⁺ levels (M) were done as described in MATERIALS AND METHODS. The results were expressed relative to the levels in MSV-pCDNA3 cells. Error bars, SE of the mean of two independent determinations done in triplicates (L) and for two single independent determinations (M), respectively.