FIG. 6.

NGF withdrawal from differentiated PC12 cells further stimulated nNOS expression and activity, and nNOS activity was inhibited by COX-2 or DLC overexpression. (A and B) PC-MT, PCXII, PC-Off, and PC-DLC cells were treated with NGF for 7 days; IPTG was added to PC-MT and PCXII cells. At 0, 3, and 5 h after NGF withdrawal, nNOS expression was analyzed by Western blotting, and activity was measured as described in Materials and Methods. Values are means ± SD of three experiments. (C) Cell lysates from differentiated PC-MT, PCXII, PC-Off, or PC-DLC cells incubated with (+) or without (−) anti-NGF antibody for 6 h were analyzed for nNOS dimerization by SDS-PAGE under low-temperature conditions as described previously (31), followed by Western blot analysis using nNOS antibody. The dimer/monomer ratio was determined by densitometrically scanning of both nNOS bands on autoradiogram.