Fig. 2.
Effects of culture medium on iNOS expression and NO production in the course of macrophage activation in F-12 or DMEM. (A) Western blot analysis of iNOS protein and β-actin using total protein extracts of JA-4 cells, cultured in F-12 or DMEM, in the presence or absence of IFN-γ (10 units/mL) and/or LPS (100 ng/mL). (B) Relative amount of iNOS protein. The results in (A) are quantitatively shown as values relative to those for the untreated cells in F-12 after normalization as to the amount of β-actin in each sample. Densitometry data are expressed as means±S.D. for three independent experiments. *p<0.05 between indicated pairs of F-12 and DMEM. (C) Western blot analysis of iNOS protein in total protein extracts of JA-4 cells treated with LPS+IFN-γ in either F-12 or DMEM at 37 °C for the times indicated on the abscissa. (D) Time-course of iNOS protein induction in F-12 and DMEM. The results in (C) are quantitatively shown as values relative to those for the untreated cells in F-12 at 0 h after normalization as to the amount of β-actin in each sample. Densitometry data are expressed as means±S.D. for three independent experiments. *p<0.05 between indicated pairs of F-12 and DMEM. (E) in the culture supernatants of macrophages after incubation with LPS+IFN-γ for the indicated times. was estimated by the Griess reagent assay. The results are shown as means±S.D. for three independent experiments. (F) Expression of iNOS mRNA during macrophage activation in F-12 and DMEM. mRNA was extracted and quantitated by qRT-PCR, as described in the text. The results are relative to those for the untreated cells in F-12 at 0 h after normalization as to the internal control, GAPDH mRNA. The results are expressed as means±S.D. for three independent experiments. *p<0.05 between indicated pairs of F-12 and DMEM.