Effects of ebselen or oxLDL on MAPK phosphorylation and expression of MAPK in mice lesions.

<p><b>A,</b> After pre-incubation for 3 days with 10 ng/ml MCSF, peritoneal macrophages were incubated for 5 min with 10 µg/ml oxLDL with or without MCSF or MCSF with or without ebselen. Cellular protein was extracted and protein samples (0.4 mg/ml) were analyzed by Western blot with specific antibodies: anti-phosphorylated MEK1/2 or anti-MEK1/2 (<b>A,</b> upper panel<b>,</b> right), anti-phosphorylated ERK1/2 or anti-ERK1/2 (<b>A,</b> middle panel<b>,</b> right) or anti-phosphorylated p90RSK or anti-RSK1/2/3 (<b>A,</b> lower panel, right) antibodies (representative experiments). ß-Actin or Actin were used as control. Quantitative results were calculated by band densitometry with the intensity of phosphorylated MEK1/2, ERK1/2, p90RSK normalized to total MEK1/2, ERK1/2, RSK1/2/3 (<b>A,</b> upper, middle and lower panel, left). Data represent mean ± SD of 5–7 separate experiments. * p<0.05 or ** p<0,01 above the histogram indicate statistically significant differences between the different genotypes and below the histogram compared with cells without treatment of MCSF or oxLDL. <b>B,</b> expression of phosphorylated MEK1/2 and ERK1/2 in parallel with staining of macrophages and SMCs in sequential sections of the aortic arch of both GPx-1^−/−ApoE^−/− (upper panels) and ApoE^−/− (lower panels) mice. There is more pronounced expression of phosphorylated ERK1/2 and MEK1/2 both in macrophages and SMCs of GPx-1^−/−ApoE^−/− compared with ApoE^−/− mice. <b>C</b>, representative double immunohistochemical staining for p-p90RSK (nuclei, brown), macrophages or SMCs (red) in ApoE^−/− mice demonstrating expression of p-p90RSK in macrophages rather than in SMCs (arrowheads). The vessel lumen is to the upper left-hand corner. The demarcation between intima and media is indicated by arrowheads.