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Fig. 2. Quantification of the Na+/K+-ATPase subunits in the plasma membrane and the whole cell fraction of HeLa cells. The amount of the 
1-, β1- and β3-subunits in the whole cell lysate and in the plasma membrane of HeLa cells at 80% confluence was quantified by immunoblot analysis using isoform-specific antibodies. The detailed procedures of cell-surface protein labeling and purification are described in supplementary material Fig. S1. (A) The total HeLa cell lysate (total) and the purified cell-surface proteins (surface) were subjected to SDS-PAGE and immunoblot analysis using the isoform-specific antibodies (1, β1 and β3) described in Fig. 1C. Note that the cell-surface fraction contains only matured forms of the β-subunits, whereas the total cell lysate contains the molecules with various sugar modifications. The positions of the molecular size markers (in kDa) are indicated on the right. Three different amounts of the samples (1, 5 and 10 µl) were subjected to the analyses to obtain a linear relationship between the amount of protein and the intensity of the immunoreactive signal on the immunoblot. The surface and total samples were blotted to the same membrane filter and subjected to exactly the same procedure to avoid experimental error. (B) A summary of the quantitative analyses. The immunoreactive bands in A were quantified, avoiding signal saturation. To compare the ratio of three subunits in each fraction, the band intensity was multiplied by the mean value of the antibody `sensitivity ratio' obtained in Fig. 1C (1:9.6:7.2 for 1:β1:β3). To compare the surface and total samples, the difference in the sample volume (50 µl surface and 500 µl total) was considered. The intracellular fraction of each subunit was obtained by subtracting the surface amount from the total amount. The surface and the intracellular fractions of 1-, β1- and β3-subunits are represented with the total amount of 1 as 1. The values in the figure were the mean values obtained from 4-5 independent experiments.
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