Figure 3. Inhibition of flippase activity by NEM reveals involvement of flippase activity in the regulation of surface PS.
A. Low surface PS cells were either treated with NEM or left untreated, incubated with NBD-PS for indicated time periods and subjected to BSA extraction and sodium dithionite treatment. % nonextractable NBD-PS (after BSA extraction and sodium dithionite treatment) represents internalized NBD-PS, indicative of flippase activity B. Flippase activity was inhibited by use of NEM in low surface PS cell lines and surface PS levels were measured by annexin V FITC binding assay, by flow cytometry. The graph shows annexin V FITC fold change compared to mock treated cells. C. High surface PS cells were either treated with NEM or left untreated and incubated with NBD-PS for indicated time periods and subjected to BSA extraction and sodium dithionite treatment. % nonextractable NBD-PS (after BSA extraction and sodium dithionite treatment) represents internalized NBD-PS, indicative of flippase activity D. Flippase activity was inhibited by use of NEM in the high surface PS cell lines and surface PS levels were measured by annexin V FITC binding assay by flow cytometry. The graph shows annexin V FITC fold change compared to mock treated cells.