Induction of cGMP and eNOS phosphorylation in TrHBMECs. (A) The cGMP levels were increased 3-fold by EPO (▪) at 21% O₂ and up to 10-fold by EPO at 2% O₂ as early as 15 minutes following EPO stimulation compared with the no EPO control (□) (^*P < .05; ^***P < .001). The eNOS inhibitor, L-NAME (▨), inhibited EPO induction of cGMP in these cells. (B) Western blot analysis showed induction of eNOS protein and eNOS phosphorylation at serine 1177 (peNOS_s1177) for cells cultured with EPO at 2% O₂. At 60 minutes, the increase in band intensity of eNOS is 2.5-fold and of peNOS_s1177 is 5.3-fold compared with 0 minutes, giving an overall increase of 2-fold in the peNOS_s1177/eNOS ratio. Error bars represent SD.