title: Monitoring RhoGTPase Activity in Leukocytes Using Classic "Pull-Down" Assays.
abstract: Inflammation is a complex process involving the contribution of leukocytes and blood vessels, which collectively aim to restore homeostasis following injury to the body. Leukocytes are essential front-line responders to infectious or noninfectious injury and can be deployed within minutes of sensing damage. A typical inflammatory response leads to the exit of circulating leukocytes into the surrounding extravascular space, which follows a series of increasingly adhesive events - collectively termed the "multistep adhesion cascade." The Ras homology (Rho) family of small GTPases (RhoGTPases) are intracellular proteins involved in translating extracellular signals into cellular behavior, such as adhesion and migration. This chapter focuses on how to prepare, perform, and monitor RhoGTPase activation assays using classic pull-down assays. Although this chapter focuses on RhoGTPase signaling downstream of L-selectin clustering, the methods outlined here can be applied to analyzing RhoGTPase activity in response to stimulating other surface receptors.
authors.affiliation: Cytoskeleton/Membrane Signalling Research Group, Cardiovascular Division, King's College London, 125 Coldharbour Lane, London, SE5 9NU, UK.
authors.forename: Marouan
authors.initials: M
authors.lastname: Zarrouk
authors.affiliation: Cytoskeleton/Membrane Signalling Research Group, Cardiovascular Division, King's College London, 125 Coldharbour Lane, London, SE5 9NU, UK.
authors.forename: David
authors.initials: D
authors.lastname: Killock
authors.affiliation: Cytoskeleton/Membrane Signalling Research Group, Cardiovascular Division, King's College London, 125 Coldharbour Lane, London, SE5 9NU, UK.
authors.forename: Izajur
authors.initials: I
authors.lastname: Rahman
authors.affiliation: Cytoskeleton/Membrane Signalling Research Group, Cardiovascular Division, King's College London, 125 Coldharbour Lane, London, SE5 9NU, UK.
authors.forename: Jessica
authors.initials: J
authors.lastname: Davies
authors.affiliation: Cytoskeleton/Membrane Signalling Research Group, Cardiovascular Division, King's College London, 125 Coldharbour Lane, London, SE5 9NU, UK. aleksandar.ivetic@kcl.ac.uk.
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Ivetić
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D007249:Inflammation / Q000378:metabolism; D019041:L-Selectin / Q000378:metabolism; D007962:Leukocytes / Q000378:metabolism*; D051379:Mice; D015398:Signal Transduction / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D019041:L-Selectin; D020741:rho GTP-Binding Proteins
keywords: Cdc42; Cytoskeleton; L-selectin; Leukocyte; Lymphocyte; Pull-down assay; Rac1; RhoA