Rapid parallel flow cytometry assays of active GTPases using effector beads.
We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.
Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA; Cancer Center, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA; Center for Infectious Diseases and Immunity, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. Electronic address: buranda@unm.edu.
Tione
T
Buranda
Soumik
S
BasuRay
Scarlett
S
Swanson
Jacob
J
Agola
Virginie
V
Bondu
Angela
A
Wandinger-Ness
D000818:Animals; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008863:Microspheres; D059010:Single-Cell Analysis; D013997:Time Factors; D014709:Vero Cells
D020558:GTP Phosphohydrolases
Cdc42; Hantavirus; Integrins; Rab GTPases; Rac; Rho