Extended Data Fig. 6.

BSA back extraction lipid scrambling assays. a, Schematic for the BSA back extraction-based lipid scrambling assay. NBD-lipids are preferentially extracted by fatty acid-free BSA. Upon extraction, the fluorescence of BSA-bound NBD-lipids is reduced by ~ 50%. b, The results with reconstituted liposomes and ATG9A-containing proteoliposomes. 1.5 mg/ml BSA was added to each reaction at t = 100s. BSA extracts NBD-lipids only from the outer leaflet of the membranes. Thus, in the absence of scrambling, ~50% of NBD-lipids on the outer leaflet lose their fluorescence by ~50%, reducing the total fluorescence by ~25%. In the presence of ATG9A, an additional ~15% of fluorescence was lost upon BSA addition, which indicates that NBD-lipids that were originally in the inner leaflet were exposed to BSA upon flipping. Source data are available online.