Phosphorylation of exogenous receptors in HEK293 cells stably expressing FLAG-tagged human β2-AR was quantified by autoradiography. At 1 day before the assay, the cells were seeded into 60-mm dishes. At 20 hours before performing the assay, the cells were pretreated with 100 μmol/L WNNO7 or GSNO, and 4 hours before the assay, the medium was changed into phosphate-free medium supplemented with [32P]orthophosphoric acid (100 μCi/mL) and 1% fetal bovine serum with or without 100 μmol/L WNNO7 or GSNO. At the assay, the cells were stimulated with 10 μmol/L ISO for 10 minutes and put on ice. The cells were then washed twice with ice-cold PBS and lyzed in ice-cold lysis buffer (50 mmol/L Tris-HCl [pH 7.4], 100 mmol/L NaCl, 2 mmol/L Na-orthovanadate, 10 mmol/L Na-PPi, 100 mmol/L NaF, 1 mmol/L dithiothreitol, protease inhibitors, and 1% sodium dodecyl sulfate) on ice by gently shaking each for 5 minutes. After 30 minutes, cell lysates were collected in tube and centrifuged at 20 000 g
Phosphorylation of exogenous receptors in HEK293 cells stably expressing FLAG-tagged human β2-AR was quantified by autoradiography. At 1 day before the assay, the cells were seeded into 60-mm dishes. At 20 hours before performing the assay, the cells were pretreated with 100 μmol/L WNNO7 or GSNO, and 4 hours before the assay, the medium was changed into phosphate-free medium supplemented with [32P]orthophosphoric acid (100 μCi/mL) and 1% fetal bovine serum with or without 100 μmol/L WNNO7 or GSNO. At the assay, the cells were stimulated with 10 μmol/L ISO for 10 minutes and put on ice. The cells were then washed twice with ice-cold PBS and lyzed in ice-cold lysis buffer (50 mmol/L Tris-HCl [pH 7.4], 100 mmol/L NaCl, 2 mmol/L Na-orthovanadate, 10 mmol/L Na-PPi, 100 mmol/L NaF, 1 mmol/L dithiothreitol, protease inhibitors, and 1% sodium dodecyl sulfate) on ice by gently shaking each for 5 minutes. After 30 minutes, cell lysates were collected in tube and centrifuged at 20 000 g