Figure 4. Membrane fractionation scheme.
Briefly, Atg5 KO MEFs were homogenized and the lysates were subjected to differential centrifugations with indicated g forces. The ability of each fraction to trigger T7-LC3 lipidation was examined. The 25,000×g (25k) pellet, which had the most activity, was selected and a sucrose gradient ultracentrifugation was performed to separate the 25k pellet to L (light) and P (pellet) fractions. The L fraction, which contained the majority of the activity to promote T7-LC3 lipidation, was further resolved on an OptiPrep gradient after which ten fractions from the top were collected and the lipidation activity was examined in each.