Fig. 6.

Detection of an alternative conformation of cyt c following peroxynitrite treatment in different cell models using mAb 1D3. (A) Control (PBS, Left) and peroxynitrite treated (300 µM in PBS, Right) were incubated overnight at 37 °C in DMEM. Fixed HeLa cells (a), melanoma murine cells (B16-F1, b), human fibroblast (IMR90, c), and BAECs (D) were incubated with mAb 1D3 antibody (1/50 dilution) followed by antimouse FITC-labeled conjugated antibody (green). Horizontal scale bars indicate 10 µm. (B) Purification of cyt c in nonnative conformation from oxidant-treated cells. BAECs were treated or not with peroxynitrite as in A and cell extracts affinity purified using mAb 1D3 linked to agarose. Bound proteins were eluted, run on 15% SDS–PAGE, and transferred to nitrocellulose membranes. Transferred proteins were probed with anti-cyt c mAb (7H8.2C12, Sigma) and developed with antimouse-IREDye-800–conjugated antibody (Odyssey). Commercially obtained cyt c was used as standard.