JCS022905 Supplementary Material

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• Supplemental Table S1 -

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• Supplemental Table S2 -

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• Supplemental Figure S1 -

  Fig. S1. Experimental design for labeling cell surface proteins with biotin followed by the purification with streptavidin. (A) A schematic diagram of cell-surface labeling system. HeLa cells were incubated with membrane-impermeable biotinylation reagent (1 mg/ml sulfo-NHS-SS-biotin) at 4°C for 90 minutes. To chase the labeled proteins, the cells were placed back in the normal medium and incubated at 37°C. After the chase for 0-5 hours, cells were either directly harvested or harvested after the treatment with glutathione for 15 minutes to remove biotin moieties remaining on the cell surface. The cells were then lysed and the labeled proteins were purified with streptavidin-agarose and analyzed by SDS-PAGE and subsequent immunoblotting. (B) Immunoblot analysis of biotinylated proteins. HeLa cells were labeled either with membrane-permeable biotinylation reagent (NHS-LC-biotin) or membrane-impermeable biotinylation reagent (sulfo-NHS-SS- biotin). After the labeling, the cells were immediately lysed and the labeled proteins were purified by streptavidin-agarose beads and then analyzed by immunoblot using antibodies against Na/K-ATPase α-subunit, BiP or Lamin B. Only the cell-surface protein (Na⁺/K⁺-ATPase α-subunit), but not the intracellular proteins (BiP and Lamin B), was labeled with sulfo-NHS-SS-biotin. The positions of the molecular size markers are indicated on the left. In our experimental conditions, almost all of the cell-surface proteins were biotinylated and recovered by streptavidin-agarose beads; almost no biotinylated protein was detected in the unbound fraction of the streptavidin-precipitation. (C) Immunofluorescence microscopy of the cell-surface proteins. HeLa cells on a coverslip were labeled with sulfo-NHS-SS-biotin. The cells were then fixed with 4% paraformaldehyde for 10 minutes at room temperature immediately after the labeling (top) or after the 3-hour chase in the normal culture medium (bottom). The cells were then incubated with Texas-Red-conjugated Streptavidin (GE healthcare) in the presence of 5% normal goat serum and 0.1% Triton X-100. Microscopic observation under a confocal laser scanning microscopy (Zeiss, PASCAL5) demonstrated that only the plasma membrane was stained immediately after the biotinylation, whereas some intracellular signal could be detected after three hours’ chase period due to the endocytosis of the labeled proteins.

• Supplemental Figure S2 -

  Fig. S2. Secondary structure prediction and hydrophobicity plot of the human Na⁺/K⁺-ATPase β-subunits suggest a possible cell-cell adhesion motif in the extracellular region. Secondary structures of β1-, β2-, β3- and β4-isoforms of the human Na⁺/K⁺-ATPase were predicted by Predator (Frishman and Argos, 1997). Hydropathy plots of these subunits were performed using Kyte-Doolitle’s hydropathy scores (window size is 11). The predicted α-helix, β-sheet and transmembrane regions are indicated over the hydrophobicity plot. Hydrophobic beta sheets are indicated by asterisks.