Accurate and reproducible measurements of RhoA activation in small samples of primary cells.
Rho GTPase activation is essential in a wide variety of cellular processes. Measurement of Rho GTPase activation is difficult with limited material, such as tissues or primary cells that exhibit stringent culture requirements for growth and survival. We defined parameters to accurately and reproducibly measure RhoA activation (i.e., RhoA-GTP) in cultured primary keratinocytes in response to serum and growth factor stimulation using enzyme-linked immunosorbent assay (ELISA)-based G-LISA assays. We also established conditions that minimize RhoA-GTP in unstimulated cells without affecting viability, allowing accurate measurements of RhoA activation on stimulation or induction of exogenous GTPase expression.
Nini
Lylia
L
Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Dagnino
Lina
L
D000818:Animals; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D051379:Mice; D011933:Reagent Kits, Diagnostic; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D011933:Reagent Kits, Diagnostic; D020742:rhoA GTP-Binding Protein