Programme en Biologie Cellulaire et Moléculaire, Faculté de Médicine Université Laval, Québec, Québec, Canada.
Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec-Université Laval, Axe de Réproduction, Santé de la Mère et de l'enfant, Québec, Québec, Canada.
PLoS One. 2022 Jul 1;17(7):e0268848. doi: 10.1371/journal.pone.0268848. eCollection 2022.
The Rho family Guanine nucleotide exchange factor (GEF) ARHGEF17 (also known as TEM4) is a large protein with only 3 annotated regions: an N-terminal actin-binding domain, a Rho-specific dbl homology (DH)- pleckstrin homology (PH) type GEF domain and a seven bladed β propeller fold at the C-terminus with unknown function. TEM4 has been implicated in numerous activities that rely on regulation of the cytoskeleton including cell migration, cell-cell junction formation and the spindle assembly checkpoint during mitosis. Here we have assessed the specificity of a TEM4 polyclonal antibody that has been commonly used as a Western blotting and immunocytochemistry probe for TEM4 in mammalian cells. We find that this antibody, in addition to its intended target, cross-reacts with the Nuclear Mitotic Apparatus Protein 1 (NuMA) in Western blotting and immunoprecipitation, and detects NuMA preferentially in immunocytochemistry. This cross-reactivity, with an abundant chromatin- and mitotic spindle-associated factor, is likely to affect the interpretation of experiments that make use of this antibody probe, in particular by immunocytochemistry and immunoprecipitation.
Rho 家族鸟嘌呤核苷酸交换因子（GEF）ARHGEF17（也称为 TEM4）是一种大型蛋白质，仅具有 3 个注释区域：N 端肌动蛋白结合结构域、Rho 特异性二结构域同源（DH）-pleckstrin 同源（PH）型 GEF 结构域和 C 端具有未知功能的七个叶片β 桨叶折叠。TEM4 已被牵连到许多依赖于细胞骨架调节的活动中，包括细胞迁移、细胞-细胞连接形成和有丝分裂期间的纺锤体组装检查点。在这里，我们评估了一种 TEM4 多克隆抗体的特异性，该抗体通常被用作哺乳动物细胞中 TEM4 的 Western blot 和免疫细胞化学探针。我们发现，这种抗体除了其预期的靶标外，还在 Western blot 和免疫沉淀中与核有丝分裂器蛋白 1（NuMA）发生交叉反应，并且在免疫细胞化学中优先检测到 NuMA。这种与丰富的染色质和有丝分裂纺锤体相关的交叉反应，可能会影响使用这种抗体探针的实验的解释，特别是通过免疫细胞化学和免疫沉淀。
