The changes in the phosphorylation of eNOS after one or two GH challenges. GH was administrated first at 0 min and again time at 75 min to MDBK cell cultures with or without AKT inhibitor pre-treatment. Whole cell extracts were prepared at the time point 0, 15, 75, 90 and 150 min after first GH challenge. Western blots using the antibodies against phospho-eNOS and phospho-AKT were performed. Panel A: The eNOS phosphorylation after one GH challenge. Panel B: The eNOS phosphorylation with two GH challenges, with or without AKT inhibitor pre-treatment for 30 min. Western blot with phospho-AKT (Thr308) was used to monitor the effectiveness of the AKT inhibitor. Loading control: Identical gel stained with SimplyBlue™ (Invitrogen). Panel C shows the quantitation of eNOS phosphorylation (Ser1177) after GH treatment. 1 GH: the quantitation for one GH challenge; 2 GH: quantitation for two GH challenge; AKTi + GH: AKT inhibitor pretreated for 30 min before GH challenge; *: First GH challenge, **: second GH challenge.