Fig. 6.

ARA1 is necessary for CD73 shedding. An anti-His antibody, recognizing His tagged recombinant CD73, was used to pull down CD73 and its associated proteins. Co-immunoprecipitate from mutant CD73-modified Cd73^−/− RPE cells was analyzed by SDS-PAGE (a). The CD73-associated protein was fractioned and subjected to amino acid sequencing of its N terminal; the sequencing result was blasted (b). The pull-down protein was identified by western blotting (c). Ara1 mRNA expression in LPS- and TNF-α-treated RPE cells was determined by qPCR (d). CD73 shedding in Ara1^−/− RPE was tested by flow cytometry (e). a SDS-PAGE result of the co-immunoprecipitate from mutant CD73-modified Cd73^−/− RPE. b Amino acid sequencing and BLAST results of fractioned CD73-associated protein. c Western blot identification of CD73-associated protein. d Ara1 mRNA expression in RPE cells tested by qPCR (n = 6). e Flow cytometry analysis of CD73 shedding in Ara1^−/− RPE