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. 2011 Jun 10;286(33):29098–29107. doi: 10.1074/jbc.M111.240127

FIGURE 3.

FIGURE 3.

C908A mutation of eNOS prohibits thiyl radical formation and uncouples eNOS. a, immunoblotting of WT eNOS and eNOS C908A to measure protein radical formation. The upper panel is eNOS protein radical formation trapped by DMPO, when eNOS is uncoupled, and immunoblotting against anti-DMPO antibody. Lane 1 is protein radical formation from WT eNOS and trapped by DMPO. Lane 2 is the same as in lane 1 with the addition of Mn-SOD. Lane 3, as in lane 1, eNOS C908A and trapped DMPO. Lane 4 is same as in lane 3 with the addition of Mn-SOD. The lower panel shows an immunoblot for eNOS. b, EPR Fe-MGD NOS activity determination. The top spectrum is the NO generated from WT eNOS. The middle spectrum is the NO generated from eNOS C908A. The bottom spectrum is the NO generated from eNOS C908A plus Mn-SOD. c, determination of O2˙̄ generation from WT eNOS and eNOS C908A by DEPMPO EPR spin-trapping. The left panel is the O2˙̄-DEPMPO EPR spectra of WT eNOS. The right panel is the O2˙̄-DEPMPO EPR spectra of eNOS C908A. All of the experiments were performed at least in triplicate.