In this article we describe the use of pharmacological and genetic tools coupled with immunoblotting (Western blotting) and targeted mass spectrometry to quantify immune signaling and cell activation mediated by tyrosine kinases. Transfer of the ATP γ phosphate to a protein tyrosine residue activates signaling cascades regulating the differentiation, survival, and effector functions of all cells, with unique roles in immune antigen receptor, polarization, and other signaling pathways. Defining the substrates and scaffolding interactions of tyrosine kinases is critical for revealing and therapeutically manipulating mechanisms of immune regulation. Quantitative analysis of the amplitude and kinetics of these effects is becoming ever more accessible experimentally and increasingly important for predicting complex downstream effects of therapeutics and for building computational models. Secondarily, quantitative analysis is increasingly expected by reviewers and journal editors, and statistical analysis of biological replicates can bolster claims of experimental rigor and reproducibility. Here we outline methods for perturbing tyrosine kinase activity in cells and quantifying protein phosphorylation in lysates and immunoprecipitates. The immunoblotting techniques are a guide to probing the dynamics of protein abundance, protein–protein interactions, and changes in post-translational modification. Immunoprecipitated protein complexes can also be subjected to targeted mass spectrometry to probe novel sites of modification and multiply modified or understudied proteins that cannot be resolved by immunoblotting. Together, these protocols form a framework for identifying the unique contributions of tyrosine kinases to cell activation and elucidating the mechanisms governing immune cell regulation in health and disease.
Bibliographical noteFunding Information:
Protocols were developed for projects with grants from the National Institutes of Health award numbers T32DA007097 (BFB) and R01AR073966, R03AI130978, T32AR007304, and F32AI082926 and from the University of Minnesota Foundation Equipment Award E-0918-01 (TSF).
Protocols were developed for projects with grants from the National Institutes of Health award numbers T32DA007097 (BFB) and R01AR073966, R03AI130978, T32AR007304, and F32AI082926 and from the University of Minnesota Foundation Equipment Award E‐0918‐01 (TSF).
© 2020 The Authors.
- Western blot
- protein tyrosine kinase
- quantitative immunoblot
- targeted mass spectrometry
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't