Figure 4.
SDS-PAGE and Western blot analysis of Glu-6-PDH treated with HNE. Glu-6-PDH (2.5 mg/ml) was incubated with HNE (5.0 mM) for 0, 0.5, 1.0, 2.0, and 4.0 h at 37°C as detailed in Materials and Methods. Samples were loaded onto two 10% polyacrylamide gels (2.0 μg/well). One gel of the series was stained with Coomassie blue for evaluation of the molecular weight distribution of HNE-treated Glu-6-PDH (A). The other gel was electroblotted onto nitrocellulose membrane (0.45 μm) for Western blot analysis with immunopurified anti-NAL2HNEf (1/2,000) as primary antibody (B). Antibody binding was visualized after exposure of the blot to a chemiluminescent substrate (Materials and Methods).