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Updated: Jul 9, 2025
Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Protocol for cell-based screening assay to measure ERK1/2 phosphorylation as a readout for complement receptor
Xaria X Li1, Trent M Woodruff1
1School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St Lucia, QLD 4072 Australia.
Abstract:
The complement receptors C3aR and C5aR1 are promising therapeutic targets. Here, we present a protocol to screen the effects of different agonists and antagonists on these receptors in vitro, using phosphorylated extracellular signal-regulated kinase (ERK) as a readout. We describe steps for isolating human monocyte-derived macrophages, culturing and preparing Chinese hamster ovary cells stably expressing human C5aR1 or C3aR, performing pharmacological assays, and detecting phospho-ERK1/2 in the cell lysate. This protocol can also be performed using other cell lines. For complete details on the use and execution of this protocol, please refer to Li et al. (2020)1 and Li et al.2.
Insights
This study details a new in vitro protocol to screen complement receptor modulators. The method uses phospho-ERK as a readout for C3aR and C5aR1 activity in immune cells and cell lines.
Area of Science:
- Immunology
- Pharmacology
Background:
- Complement receptors C3aR and C5aR1 are key therapeutic targets.
- Understanding their modulation is crucial for developing new treatments.
Purpose of the Study:
- To present a detailed protocol for screening agonists and antagonists of C3aR and C5aR1 in vitro.
- To establish a reliable method using phosphorylated extracellular signal-regulated kinase (p-ERK) as a readout.
Main Methods:
- Isolation of human monocyte-derived macrophages.
- Culture and preparation of Chinese hamster ovary cells expressing human C3aR or C5aR1.
- Pharmacological assays and detection of p-ERK1/2 in cell lysates.
Main Results:
- The protocol enables effective screening of receptor modulators.
- Phospho-ERK1/2 levels serve as a quantifiable readout for receptor activity.
- The method is adaptable to various cell lines.
Conclusions:
- This protocol provides a robust platform for studying complement receptor pharmacology.
- It facilitates the discovery and characterization of novel therapeutic agents targeting C3aR and C5aR1.
