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. 2019 Mar 15;36(7):1060–1079. doi: 10.1089/neu.2018.5986

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© Volodymyr Gerzanich et al., 2018; Published by Mary Ann Liebert, Inc.

This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.

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FIG. 1. — KIR6.2 antibody validation. (A) Whole–cell lysate (WL) or anti-myc immunoprecipitate (IP) were prepared from COS-7 cells transfected with myc-KIR6.2 or empty vector; immunoblot with anti-myc antibody showed no bands with expression of empty vector (lanes 1, 3), and showed KIR6.2 monomers and multimers with KIR6.2 expression (lanes 2, 4); immunoblot with GTX80493 anti-KIR6.2 antibody showed a single faint nonspecific band in WL immunoblot of empty vector (lane 5), and showed KIR6.2 monomers and multimers with KIR6.2 expression (lanes 6, 8); representative of three experiments. (B) Whole lysate was prepared from COS-7 cells transfected with myc-KIR6.2 or empty vector; immunoblot with anti-myc antibody showed no bands with expression of empty vector (lane 1), and showed KIR6.2 monomers and multimers with KIR6.2 expression (lane 2); immunoblot with sc-11228 anti-KIR6.2 antibody showed multiple nonspecific bands with expression of empty vector (lane 3), and no KIR6.2 with expression of KIR6.2 (lane 4); immunoblot with sc-11226 anti-KIR6.2 antibody showed multiple nonspecific bands with expression of empty vector (lane 5), and showed KIR6.2 multimers with expression of KIR6.2 (lane 6); representative of three experiments. (C) Whole lysate was prepared from COS-7 cells transfected with empty vector, or myc-KIR6.1 or myc-KIR6.2; immunoblot with anti-myc antibody showed no bands with expression of empty vector, and showed KIR6.1 or KIR6.2 multimers with KIR6.1 or KIR6.2 expression, as indicated; immunoblot with GTX80493 anti-KIR6.2 antibody showed a single faint nonspecific band with empty vector and with KIR6.1 expression, and showed multimers with KIR6.2 expression, as indicated; representative of three experiments. (D) Combined in situ hybridization and double immunolabeling of the same tissue section from human brain after TBI. Upper panel: superimposed double immunolabeling for KIR6.2 (GTX80493, GeneTex; green) and glial fibrillary acidic protein (GFAP; red) showed astrocytes that express KIR6.2 (asterisks); in the same field, other cells identified by 4′,6-diamidino-2-phenylindole staining of nuclei showed no immunoreactivity for GFAP or KIR6.2 (red circles). Lower panel: in situ hybridization for Kcnj11 in the same tissue section showed positive signal within the same astrocytes that express KIR6.2 protein (asterisks), and no signal in cells that express no KIR6.2 protein (circles); representative of five experiments. (E) Double immunolabeling for KIR6.2 using APC-020 (Alomone Labs; green) and GFAP (red) in tissues from the same case as in (D) confirmed the specificity of the anti-KIR6.2 antibody, GTX80493. (F) Double immunolabeling for KIR6.2 (GTX80493, GeneTex; green; green) and GFAP (red) in control (uninjured) cortical tissue showed that GTX80493 exhibited no immunoreactivity when used to label control GFAP+ tissues.