PGEC and RBC lysate preparation

PGEC were cultured to confluence, washed twice with cold PBS (Hyclone, Logan, UT) and scraped in RIPA lysis buffer with protease inhibitor cocktail (Santa Cruz Biotechnology, Dallas, TX). PGEC lysates were freeze-thawed once and centrifuged at 10,000 × g for 10 min at 4 °C. Supernatants were stored at -20 °C until analyzed for levels of arginase 1. Protein concentration was assessed by the Bicinchoninic assay (Sigma-Aldrich).

For determination of intracellular RBC arginase 1, RBC lysates were prepared. Briefly, whole blood from healthy adult volunteers was centrifuged at 150 × g for 15 min at rt. The RBC pellet was then washed four times in large amounts of PBS without calcium and magnesium (Hyclone), followed by centrifugation at 500 × g at rt. After each centrifugation, the supernatant was fully removed together with the supernatant-pellet interface to remove white blood cells. The RBC solution at 150 µL was then incubated with dH₂0 1350 µL for 20 min with gentle rocking at 4 °C, followed by centrifugation at 2500 × g for 15 min at 4 °C. Protein concentration was determined by the Bicinchoninic assay and supernatants stored at − 20 °C until used for measurement of arginase 1 levels.