Fig. 2.
Effects of globular adiponectin on ethanol-induced Nox2 and p22phox expression in RAW 264.7 macrophages. (A) Cells were incubated with gAcrp (0.1 μg/ml) for the indicated time periods. Nox2 mRNA expression was analyzed by qRT-PCR as described in Materials and Methods. Values are represented as fold increase relative to controls and are expressed as mean ± S.E.M. (n = 4). *P < 0.05 compared with cells not treated with gAcrp. (B) Cells were treated with gAcrp (0.1 μg/ml) for the indicated time periods. Nox2 protein expression levels were analyzed by Western blot analysis as described in Materials and Methods. Representative image from four independent experiments is shown along with β-actin for internal loading control. Band intensities were quantified by densitometric analysis and normalized by β-actin. Values are represented as fold changes relative to controls and are expressed as mean ± S.E.M. (C) Cells were treated with gAcrp (0.1 μg/ml) for the indicated time periods. p22phox mRNA expression was analyzed by qRT-PCR as described previously. Values shown are represented as mean ± S.E.M. (n = 3). *P < 0.05 compared with cells not treated with ethanol. (D) Cells were pretreated with gAcrp (0.1 μg/ml) for 18 hours, followed by 100 mM of ethanol for an additional 24 hours. Nox2 mRNA expression level was assessed by qRT-PCR as described previously. Values are presented as mean ± S.E.M. (n = 4). *P < 0.05 compared with cells not treated with ethanol; #P < 0.05 compared with cells treated with ethanol. (E) Cells were pretreated with 0.1 μg/ml of gAcrp for 18 hours, followed by ethanol for additional 24 hours. Nox2 protein was measured by Western blot analysis as described previously. Images are representative of three separate experiments that showed similar results. Band intensities were quantified by densitometric analysis and normalized by β-actin. Values are represented as fold changes relative to control and are expressed as mean ± S.E.M. (F) Cells were treated with gAcrp (0.1 μg/ml) for 18 hours, followed by incubation with 100 mM ethanol (100 mM) for 24 hours. p22phox mRNA expression was measured as described previously. Values are presented as mean ± S.E.M. (n = 4). *P < 0.05 compared with controls; #P < 0.05 compared with cells treated with ethanol.