FIGURE 2.

Levels of Sms expression and cell-surface SM in Sms-reconstituted cells. A, cell lysates (22 μg of protein/lane) from SM-deficient mouse embryonic fibroblasts (ZS cells) and Sms1- or Sms2-expressing ZS cells (ZS/Sms1 and ZS/Sms2 cells, respectively) were subjected to Western blotting with the anti-V5 antibody to confirm their expression levels. Western blotting was also performed with the anti-GAPDH antibody for the loading control. IB, immunoblot. B, amount of SM on the cell surface was determined by the sensitivity of lysenin. Cell numbers were determined by the WST-1 cell proliferation reagent after ZS (filled square), ZS/Sms1 (open triangle), and ZS/Sms2 cells (filled circle) were cultivated with various amounts of lysenin (0–10 μg/ml). Values represent the mean ± S.D. from three independent experiments. C, amount of SM on the cell surface was also determined using a probe for SM (EGFP-lysenin). Each cell was stained with 3 μg/ml EGFP-lysenin and analyzed by flow cytometry. Ten thousand cells were measured in each sample. The filled histogram represented cells that bound EGFP-lysenin. In contrast, the open histogram indicates cells without staining for EGFP-lysenin. One representative experiment is shown, and similar results were obtained in three independent experiments.