Mechanisms of abnormal NO synthase activity. Lung homogenate analysis is shown for 4 to 7 mice per group. (A) Lung NO synthase activity in the presence of calcium (constitutive NOS activity in the lung, the vast majority of which is eNOS) was decreased in sickle mice. (B) Calcium-independent citrulline formation (iNOS activity) did not differ between groups. (C) Western blot under nondenaturing conditions demonstrated 280 kDa eNOS dimer (active form) and 140 kDa eNOS monomer, which is inactive.^53,54  Hemizygous mice had slightly more eNOS dimer than monomer, but sickle mice had almost completely lost dimerized eNOS. Positive controls show eNOS dissociated completely to monomeric form by boiling. Images were acquired by scanning the gel (Microtek [Carson, CA] i700 flatbed scanner with a transparency adapter) and saved without processing as a “tiff” image and then converted to “jpg” using Adobe Photoshop CS. (D) Lung nitrotyrosine, evidence of NO scavenging by reaction with superoxide or nitrite myeloperoxidase, was elevated in sickle mice and nitrotyrosine when measured by colorimetric assay (Oxis International) following the manufacturer's specifications. Statistically significant results indicated as follows: *P <.05 versus hemizygote; #P < .05 versus mice receiving transplants with wild-type marrow (BM-C). Numbers represent mean ± SEM.