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CB2 receptor antibody signal specificity: correlations with the use of partial CB2-knockout mice and anti-rat CB2 receptor antibodies.
Acta Pharmacologica Sinica ( IF 8.2 ) Pub Date : 2018-07-02 , DOI: 10.1038/s41401-018-0037-3
Hai-Ying Zhang 1 , Hui Shen 2 , Chloe J Jordan 1 , Qing-Rong Liu 3 , Eliot L Gardner 1 , Antonello Bonci 2 , Zheng-Xiong Xi 1
Affiliation  

Cannabinoid CB1 receptors are highly expressed in the brain and functionally modulate presynaptic neurotransmitter release, while cannabinoid CB2 receptors (CB2Rs) were initially identified in the spleen and regarded as peripheral cannabinoid receptors. Recently, growing evidence indicates the presence of functional CB2Rs in the brain. However, this finding is disputed because of the specificity of CB2R antibody signals. We used two strains of currently available partial CB2-knockout (CB2-KO) mice as controls, four anti-rat or anti-mouse CB2R antibodies, and mRNA quantification to further address this issue. Western blot assays using the four antibodies detected a CB2R-like band at ~40 kD in both the brain and spleen. Notably, more bands were detected in the brain than in the spleen, and specific immune peptides blocked band detection. Immunohistochemical assays also detected CB2-like immunostaining in mouse midbrain dopamine neurons. CB2R deletion in CB2-KO mice may reduce or leave CB2R-like immunoreactivity unaltered depending on antibody epitope. Antibodies with epitopes at the receptor-deleted region detected a significant reduction in CB2R band density and immunostaining in N-terminal-deleted Deltagen and C-terminal-deleted Zimmer strain CB2-KO mice. Other antibodies with epitopes at the predicted receptor-undeleted regions detected similar band densities and immunostaining in wild-type and CB2-KO mice. Quantitative RT-PCR assays detected CB2 mRNA expression using probes that targeted upstream or downstream gene sequences but not the probe that targeted the gene-deleted sequence in Deltagen or Zimmer CB2-KO mice. These findings suggest that none of the tested four polyclonal antibodies are highly mouse CB2R-specific. Non-specific binding may be related to the expression of mutant or truncated CB2R-like proteins in partial CB2-KO mice and the use of anti-rat CB2 antibodies because the epitopes are different between rat and mouse CB2Rs.

中文翻译:

CB2 受体抗体信号特异性:与使用部分 CB2 敲除小鼠和抗大鼠 CB2 受体抗体的相关性。

大麻素 CB1 受体在大脑中高度表达并在功能上调节突触前神经递质的释放,而大麻素 CB2 受体 (CB2Rs) 最初在脾脏中被鉴定并被视为外周大麻素受体。最近,越来越多的证据表明大脑中存在功能性 CB2R。然而,由于 CB2R 抗体信号的特异性,这一发现存在争议。我们使用两种目前可用的部分 CB2 敲除 (CB2-KO) 小鼠作为对照,四种抗大鼠或抗小鼠 CB2R 抗体和 mRNA 量化来进一步解决这个问题。使用四种抗体的蛋白质印迹分析在大脑和脾脏中检测到约 40 kD 的 CB2R 样条带。值得注意的是,大脑中检测到的条带多于脾脏,并且特异性免疫肽阻断了条带检测。免疫组织化学测定还检测到小鼠中脑多巴胺神经元中的 CB2 样免疫染色。CB2-KO 小鼠中的 CB2R 缺失可能会降低或保持 CB2R 样免疫反应性不变,具体取决于抗体表位。在受体缺失区域具有表位的抗体检测到 N 末端缺失的 Deltagen 和 C 末端缺失的 Zimmer 菌株 CB2-KO 小鼠的 CB2R 条带密度和免疫染色显着降低。在预测的受体未缺失区域具有表位的其他抗体在野生型和 CB2-KO 小鼠中检测到相似的条带密度和免疫染色。定量 RT-PCR 检测使用针对上游或下游基因序列的探针而不是针对 Deltagen 或 Zimmer CB2-KO 小鼠中基因缺失序列的探针检测 CB2 mRNA 表达。这些发现表明,所测试的四种多克隆抗体都不是高度小鼠 CB2R 特异性的。非特异性结合可能与部分 CB2-KO 小鼠中突变或截短的 CB2R 样蛋白的表达以及抗大鼠 CB2 抗体的使用有关,因为大鼠和小鼠 CB2R 的表位不同。
更新日期:2018-07-03
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