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.