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. 2016 Jul 27;64(11):1938–1961. doi: 10.1002/glia.23034

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© 2016 The Authors. Glia Published by Wiley Periodicals, Inc.

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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Identification of rat GPNMB protein for 9F5 antigen by using both immunoprecipitation (IP) and microsequencing. A: Lysates of rat type 1 MG were immunoprecipitated by 9F5 or control IgG and then immunoblotted with 9F5. Arrows indicate staining specific for 9F5. Asterisks indicate nonspecific staining. B: Lysates of rat type 1 MG were immunoprecipitated by 9F5, subjected to SDS‐PAGE, and then transferred to PVDF membranes. Membranes were evaluated via immunoblotting (lanes 1, 2) or staining with Coomassie Brilliant Blue R250 (CBB) (lanes 3, 4), and the major bands with estimated mol wt 50–70 kDa (lane 3, bands 1–3) were extracted and analyzed by using a microsequencer (C). Asterisks indicate nonspecific staining. Ca: Yield of phenylthiohydantoin (PTH)‐amino acid per cycle number (bands 2, 3). b: Amino acid sequence comparison of the N‐terminal amino acid sequence of bands 1–3 with rat GPNMB (AAH61725) and rat osteoactivin (NP_579832). Shading indicates the residues matched with rat GPNMB. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]