FIGURE 6.

Effect of PID on iNOS during its de novo synthesis as well as after complete assembly into dimeric iNOS in induced RAW 264.7 cells. A shows the expression levels of iNOSfl and its relative dimer-monomer content in the extracts of PID-treated and -untreated cells as determined through low temperature SDS-PAGE immunoblotting of iNOSfl. RAW 264.7 cells were induced with LPS (50 μg/ml) and IFN-γ (10 ng/ml) to express iNOSfl in the presence (A, lanes 2, 4, and 6) or absence (A, lanes 3, 5, and 7) of l-NAME. Cells induced for 10 h without PID showed discernible amounts of dimer both in the presence (A, lane 4) or absence (A, lane 5) of l-NAME. PID (10 μm) was added either along with initiation of induction and harvested after 10 h (A, lanes 2 and 3) or after 10 h of induction following which cells were incubated for 6 h with PID before being harvested (A, lanes 6 and 7). The iNOS immunoblot of the uninduced control is shown in A, lane 1. B graphically depicts the percentage dimer-monomer content as evidenced through gel filtration chromatographic analysis of the purified iNOSfl from the treated cells. C shows the corresponding NOS activities in terms of the nitrite produced in a Griess reaction assay by the iNOSfl proteins purified from the above-treated cells.