Cellular signal transduction comprises a complex series of biochemical reactions by which extracellular signals such as growth factors, hormones, cytokines, and neurotransmitters are translated into specific intracellular responses. Signal transduction is mediated by protein kinase phosphorylation cascades that culminate in the regulation of numerous cellular responses, including division, differentiation, migration, and survival. Importantly, signal relay pathways are dysregulated in human diseases, making the study of signal transduction important for both uncovering basic biology and understanding pathophysiology. Established laboratory cell culture models are useful for studying signal transduction mechanisms, but differences in sample handling procedures can introduce unwanted variability in experimental outcomes and conclusions. One such potential source of experimental variability is the introduction of fluid shear stress upon handling of tissue culture cells. Fluid shear stress triggers a wide range of cellular responses in adherent cell culture, including stimulating the production of cyclic AMP, potentiating the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), and ultimately inducing changes in the gene expression of growth and remodeling factors. Further, mechanical stress on cells is physiologically relevant to the development of many pathologies. Here, we describe a detailed protocol for cell lysis and protein extraction that minimizes shear stress induced by classical cell harvest protocols. We also highlight the impact of fluid shear stress by using immunoblotting to assess ERK pathway activation as a readout for this protocol. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Gentle cell lysis and protein extraction Basic Protocol 2: Immunoblotting for cell signaling readouts by SDS-PAGE.
Keywords: ERK1/2; SDS-PAGE; cell signaling; lysis; protein extraction; shear stress.
© 2024 Wiley Periodicals LLC.