Induction of epithelial CD73 by hypoxia. (a) Confluent T84 monolayers were exposed to normoxia (pO2 147 torr, 18 hours) or hypoxia (pO2 20 torr, 18 hours). Total RNA was isolated, and CD73 mRNA levels were determined by RT-PCR using semiquantitative analysis (increasing cycle numbers, as indicated). As shown, β-actin transcript was determined in parallel and used as an internal standard. (b) Confluent T84 monolayers were exposed to indicated periods of hypoxia (with or without actinomycin D [Act. D], as indicated), monolayers were washed, surface proteins were biotinylated, and cells were lysed. CD73 was immunoprecipitated with mAb 1E9 and resolved by SDS-PAGE, and resultant Western blots were probed with avidin-peroxidase. Representative experiments of three are shown in each case.