Fig. 4

NO is a potent inductor of HO-1 mRNA and protein expression in the RAW 264.7 macrophage cell line. (A) Adhered RAW 264.7 macrophages were treated with the NO-donating reagents GSNO (500 µM), or SpNO (350 µM) for 4 and 8 hr as indicated. Total protein (50 µg) of the cells was analyzed by immunoblotting for the presence of HO-1-specific protein. A Ponceau S staining of the same gel is shown as loading control. One representative experiment is shown. (B and C) RAW 264.7 cells were stimulated with LPS (10 µg/ml)/IFN-γ (100 U/ml) in the presence or absence of L-NMMA (2 mM). NO-production of the cells after 8 hr in the presence of these reagents was assessed by measuring nitrite accumulation in the cell culture supernatants as indicated (B). Data are expressed as percent of unstimulated control ± SD (n = 3). *p < 0.05 compared with the conditions as indicated by the bracket. (C) Cells were treated as indicated. Twenty micrograms of total cellular RNA (C, upper panel), or 50 µg of total protein lysate (C, lower panel) were analyzed for the presence of HO-1 expression at indicated time points by RNase protection assay or Western blot analysis, respectively. HO-1 specific signals were indicated by arrows. Expression of GAPDH mRNA (C, upper panel), or Ponceau S staining (C, lower panel) of the membrane, respectively, were shown as loading controls.