Fig. 5

Treatment with STEP mimetic alleviates post-ischemic inflammatory response in neurons of SHR rats. (A) Diagram of STEP₆₁ indicating the positions of the phosphatase domain, putative proteolytic sites (PEST), transmembrane domain (TM), polyproline rich regions (PP), kinase interacting motif (KIM), kinase specificity sequence (KIS) and known phosphorylation sites. The STEP mimetic (TAT-STEP-myc peptide) was generated from STEP₆₁. The peptide was rendered cell-permeable by fusion to the 11 amino acid protein transduction domain (TAT) of the human immunodeficiency virus-type I at the N-terminus and has a myc-tag at the C-terminus. The serine residue in the KIM domain was mutated to alanine to allow the peptide to bind constitutively to its substrates. The threonine and serine residues in the KIS domain were mutated to glutamic acid to render the peptide resistant to degradation. (B-L) SHR rats were subjected to MCAO (60 min) followed by reperfusion (RPF) for 6 h. TAT-STEP-myc peptide (TAT-STEP) was administered in a subset of SHR rats at the onset of reperfusion. (B) ERK MAPK phosphorylation and (G) IκB protein level were evaluated by immunoblot analysis of tissue extracts from ipsilateral cortex. Blots were re-probed with (B) anti-ERK and (G) anti-β-tubulin antibodies (lower panels). Bar graphs represent mean ± SD (n = 3). (C-F, I, J) Tissue extracts from the ipsilateral cortex were processed to measure mRNA levels of (C, D) NFκB1, (E, F) RelA and (I, J) COX-2 by quantitative real-time PCR. Bar graphs represent relative expression of NFκB1, RelA and COX-2 normalized against reference genes β-actin or HPRT (mean ± SE, n = 6). (H, K) Coronal brain sections through the ipsilateral cortex were processed for immunohistochemical staining with (H) anti-pNFκB (red) antibody, anti-NeuN antibody (green) and nuclear stain DAPI (blue); and (K) anti-COX-2 antibody (red), anti-NeuN antibody (green) and nuclear stain DAPI (blue). Arrows in the representative photomicrographs demonstrate (H) nuclear localization of pNFκB in NeuN positive cells and (K) localization of COX-2 in NeuN positive cells. (I, J). (L) PGE₂ level was measured by enzyme immunoassay using supernatants obtained from ipsilateral cortex. Bar graphs represent quantitative analysis of PGE₂ level (mean ± SD, n = 3). (B-G, I, J, L) Significant difference between means was assessed using student t-test and presented as *p < 0.05, **p < 0.01 and ***p < 0.001