MetAP2 is cleaved by calpain in a rat middle cerebral arterial occlusion (MCAO) stroke model(A) Tissue harvested at 1 hour post-reperfusion did not show a difference in the formation of a proteolytic fragment between contralateral (C) and ipsilateral (I) brain. (B) Western blotting of MCAO brain tissue harvested 24 hours post-ischemia is shown in lanes 1-8. Lanes 9 and 10 show the in vitro digestion of contralateral MCAO brain sample in the presence (+CPN) or absence (-CPN) of calpain 2. A 57 kDa fragment is clearly observed in 3 of 4 samples (I) that runs at an identical molecular weight on SDS-PAGE as calpain-treated brain homogenates (+CPN). (C) A significant increase in the novel 57 kDa calpain-mediated MetAP2 cleavage product was clear at 24 hours post-ischemia (* = p=0.02 by Students t-test, two-tailed, paired, n=4). (D) Treatment of contralateral MCAO brain samples in the presence of calpain and calpastatin indicates that the 57kDa fragment produced by calpain 2 is blocked by the only known endogenous calpain inhibitor, calpastatin. Homogenate was prepared using commercially available mouse brain (p/n MS-T004). Figure 3. PMID: 23295187.