2 articles published in JoVE
Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers H.S. Jeffrey Man1,2, Aravin N. Sukumar1,2, Kyung Ha Ku2,3, Michelle K. Dubinsky1,2, Noeline Subramaniam1,2, Philip A. Marsden1,2,3,4 1Institute of Medical Science, University of Toronto, 2 Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.
Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates Faiza Waheed1,2, Pamela Speight1,2, Qinghong Dan1,2, Rafael Garcia-Mata3, Katalin Szaszi1,2 1Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, 2Department of Surgery, University of Toronto, 3Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.