Abstract
Monitoring cellular signaling events can help better understand cell behavior in health and disease. Traditional immunoassays to study proteins involved in signaling can be tedious, require multiple steps, and are not easily adaptable to high throughput screening (HTS). Here, we describe a new immunoassay approach based on bioluminescent enzyme complementation. This immunoassay takes less than two hours to complete in a homogeneous “Add and Read” format and was successfully used to monitor multiple signaling pathways’ activation through specific nodes of phosphorylation (e.g pIκBα, pAKT, and pSTAT3). We also tested deactivation of these pathways with small and large molecule inhibitors and obtained the expected pharmacology. This approach does not require cell engineering. Therefore, the phosphorylation of an endogenous substrate is detected in any cell type. Our results demonstrate that this technology can be broadly adapted to streamline the analysis of signaling pathways of interest or the identification of pathway specific inhibitors.
Highlights
Monitoring cellular signaling events can help better understand cell behavior in health and disease
The NanoLuc Binary Technology (NanoBiT) system is composed of two subunits, Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT; 11 amino acid peptide), that can be expressed as fusions or chemically conjugated to target proteins of interest
The amount of total IκBα protein decreased due to its degradation after TNFα treatment and this reduction was abolished with IKK16 treatment (Fig. 3b). These results show that NanoBiT immunoassay reagents combined with an IκBα primary antibody pair can, in a simple way reveal the predicted biology of NF-κB signaling pathway upon TNFα treatment
Summary
Monitoring cellular signaling events can help better understand cell behavior in health and disease. CRISPR/Cas[9] techniques have been developed to express endogenous level of proteins by editing the cell genome and introducing small tags in frame with the target protein[4,5,6] Some of these methods can be sensitive to detect native PTM levels, they can be tedious, require multiple washing steps (non-homogeneous), or the necessary cell engineering is a requisite upfront hurdle for assay design. Only when two target proteins tagged with these subunits interact, the subunits come together to form an active enzyme and generate a bright luminescent signal in the presence of its substrate furimazine In this new immunoassay, the NanoBiT subunits are conjugated to an anti-mouse and an anti-rabbit secondary antibody (NanoBiT detecting antibodies), which recognize two primary antibodies to a single protein, one to a phosphorylated and the other to a nonphosphorylated epitope, or to two non-phosphorylated epitopes. These applications will help scientists identify and validate novel pathway specific drug therapies for the treatment of various diseases
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