Abstract
The family of Rab GTPases switch between GDP- and GTP-bound forms to interact with effectors and accessory proteins for the regulation of trafficking and signaling pathways in cells. The activation and recruitment of a specific Rab by stimulants or physiological changes can be detected and assessed by measuring the relative amount of the Rab in its active, “GTP-bound” state versus the inactive “GDP-bound” state. While GTP loading can be measured in vitro, current methods to detect the activation state of endogenous Rabs within a cellular context are limited. Here, we developed two molecular probes, based on domains of known Rab effectors, which can be used to pull down endogenous GTP-bound Rab8 from cell extracts as a measure of Rab8 activation. As a test system, we use the lipopolysaccharide (LPS) induced activation of Rab8 in mouse macrophages. The molecular probes compared for capture of GTP-bound Rab8 are derived from two Rab8 effectors, OCRL and PI3Kγ, with the former assessed as being more efficient. We describe how the OCRL-RBD probe is used to assess activation of Rab8 in cell extracts with a method that should be applicable to assessing GTP-bound Rab8 in other cell and tissue extracts.
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Acknowledgments
We wish to thank colleagues from the Itzen laboratory Max Planck Institute of Molecular Physiology, Germany) for provision of the OCRL539-901 construct, we thank Tatiana Khromykh (IMB) for help with cloning. This work was supported by fellowship (JLS:APP1176209) and grant funding (APP1101072, APP1138723, and APP1159106) from the National Health and Medical Research Council of Australia and fellowship (LL: DE180100524) funding from the Australian Research Council.
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Tong, S.J., Lucas, R.M., Xiao, Z., Luo, L., Stow, J.L. (2021). Detecting Endogenous Rab8 Activation. In: Li, G., Segev, N. (eds) Rab GTPases. Methods in Molecular Biology, vol 2293. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1346-7_4
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DOI: https://doi.org/10.1007/978-1-0716-1346-7_4
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