Fig. 7.

Lipid raft localization is necessary for CD73 shedding. Cell membrane extract was primarily isolated from differently treated RPE cells, further separated into lipid raft and non-lipid raft fractions by either detergent free (a) or detergent resistant methods (b). All the fractions were western blotting for CAV-1 as the marker of lipid rafts fractions and Na⁺/K⁺ ATPase as the marker of non-lipid fractions. The effect of mevastatinon CD73 shedding (c), CD73 lipid rafts localization (e) and ARA1 co-IP (f) were evaluated by western blotting. The amount of active MMP-9 in the medium was detected (D). a The existence of CD73 and ARA1 in lipid raft and non-lipid raft fractions, determined by western blotting. b The co-existence of CD73 and ARA1 in lipid raft fractions prepared by detergent resistant methods. c Localization of ARA1 in Cd73^−/− RPE, with or without Wt-CD73 modification. d The effect of mevastatin on CD73 shedding was determined by western blotting (upper panel). Active MMP-9 in the medium was determined by MMP-9 Biotrak activity assay (lower panel, n = 6). e The effect of mevastatin on CD73 localization, determined by western blotting. f The effect of mevastatin on the Co-IP of CD73 and ARA1