Fig. 2

Loss of basal STEP function in SHR rats is associated with sustained increase in neuronal ERK MAPK phosphorylation during ischemia and reperfusion. (A) Cortical tissue lysates from SD and SHR rats with equal amount of protein were processed for immunoblot analysis under non-reducing condition (without β-mercaptoethanol) using anti-STEP antibody to evaluate STEP dimer formation (upper panel). The same lysates were also processed for immunoblot analysis under reducing condition (with β-mercaptoethanol) using anti-STEP antibody to assess total STEP expression level (lower panel). Bar graphs represent quantitative analysis of STEP dimer formation (mean ± SD, n = 6). (B) STEP was immunoprecipitated from equal amount of cortical tissue lysates of SD and SHR rats using anti-STEP antibody and the immune complexes were processed either for tyrosine phosphatase activity assay using pNPP as a substrate or immunoblotting with anti-STEP antibody to ensure equal pull down of STEP. Bar graphs represent quantitative analysis of STEP phosphatase activity (mean ± SD, n = 6) (C) SD and SHR rats were subjected to sham surgery or MCAO (I) for the specified time periods (10 and 60 min). In some experiments 60 min MCAO was followed by reperfusion (RPF) for the specified time periods (3, 6 and 18 h). Tissue lysates with equal amount of protein from the ipsilateral cortex were analyzed using anti-phospho-ERK1/2 (pERK1/2) MAPK antibody (upper panels). Equal protein loading was confirmed by re-probing the blots with anti-ERK2 antibody (lower panels). Bar graphs represent quantitative analysis of pERK levels (mean ± SD, n = 4). (A-C) Significant difference between means was assessed by student t-test and presented as *p < 0.01, **p < 0.001 and ***p < 0.0001. (D) SD and SHR rats were subjected to sham surgery or 60 min MCAO followed by reperfusion for 6 h and then processed for immunohistochemical staining with anti-phospho-ERK1/2 (pERK - red) antibody, a neuron specific antibody, anti-NeuN (green) and nuclear stain DAPI (blue). Arrows in the representative photomicrographs demonstrate localization of phosphorylated ERK in NeuN positive cells in the ipsilateral cortex