Fferences were found in the amounts of caspase-cleaved -spectrin fragments between > 자유게시판

Fferences were found in the amounts of caspase-cleaved -spectrin fragments between any AD tissue and control. This suggests that the increased amounts of 145?50 kDa -spectrin bands detected in AD are due to the action of calpain and not caspases, which is in keeping with our analysis of these protease activities (Fig. 3a ).Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:Page 7 ofFig. 3 (See legend on next page.)Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:Page 8 of(See figure on previous page.) Fig. 3 Active calpain-1 amounts are elevated early in AD and are sustained throughout disease progression. a Representative blots of cortical homogenates from postmortem brain. Blots were probed with antibodies to detect active calpain-1 at 76 kDa and active/cleaved caspase-3 at 19 kDa. An antibody against calpastatin (CAST) was used to detect CAST holoprotein at 110 kDa, active CAST at > 25 kDa and inactive CAST at Capecitabine pathogenesis [19, 55, 69]. Cdk5 is activated when it forms a complex with one of its neuronal activators, such as p35. When cleaved by calpain, p35 yields the more stable and potent activator, p25, sustained expression of which is associated with increased tau phosphorylation and tau-associated synaptic and neuronal loss in vivo [12, 52]. Here, blots of AD brain lysates were probed with antibodies against cdk5, yieldinga band of 33 kDa, and p35 which detects both p35 (35 kDa) and p25 (25 kDa) (Fig. 4a). Quantification of these results showed no significant changes in total cdk5 protein, p35 or p25 amounts in AD brain when compared to control (Fig. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6833145 4b, c). However, when p25 was measured as a proportion of p35, we found a significant increase in the p25/p35 ratio in Braak stage III to V brain (p < 0.05 for all) compared to control (Fig. 4d), the same disease stages in which calpain activity was found to be significantly elevated. This was indicative that increased calpain-mediated p25 generation and therefore increased cdk5 activity occurs from an early stage of ADFig. 4 Changes in cdk5 and GSK-3 activities with AD progression. a Representative blots of cortical homogenates from postmortem brain. Blots were probed with antibodies against cyclin dependent kinase 5 (cdk5) to detect holoprotein at 33 kDa, p35 to detect holoprotein at 35 kDa and calpain-cleaved 25 kDa fragments (p25) at 25 kDa, total glycogen synthase kinase 3/ (totGSK3) at 47 and 51 kDa, respectively and GSK3/ phosphorylated at Ser21/9.