A commercial ARHGEF17/TEM4 antibody cross-reacts with Nuclear Mitotic Apparatus protein 1 (NuMA)

Abstract

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.

ARHGEF17/TEM4 regulates the cell cycle through control of G1 progression

“…1B)(Isokane et al, 2016). To study the function of TEM4 in mitosis, we first generated inducible HeLa-TRex cell lines expressing two independently verified shRNAs targeting TEM4 (hereafter referred to as TEM4-21 and TEM4-22) (Isokane et al, 2016; Memon et al, 2021; Ngok et al, 2013; Prifti et al, 2022; Weber et al, 2021). We initially validated the efficiency of TEM4 depletion by qPCR and detected a 50-60 % decrease in TEM4 mRNA levels 72 hours post shRNA induction (Fig.…”

“…HeLa-TREx stable cell lines expressing inducible shRNA targeting TEM4 (TEM4-21 and TEM4-22) have been previously described (Prifti et al, 2022). The same approach was used for generation of the HCT116 lines with inducible TEM4 depletion.…”

“…1A). TEM4 binds specifically and directly to dynamic, newly assembled F-actin filaments, via the ABD (Mitin et al, 2012; Prifti et al, 2022). In vitro studies indicated that the catalytic activity of TEM4 is specific for Rho over Rac1 and Cdc42 (Bagci et al, 2020; De Toledo et al, 2000; Mitin et al, 2012; Rümenapp et al, 2002a).…”