A homogeneous bioluminescent immunoassay to probe cellular signaling pathway regulation

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.

Keywords: Cell signalling; Immunological techniques; Target validation.

Fig. 1. NanoBiT cell-based immunoassay.

a Principle of NanoBiT cell-based immunoassay. Phosphorylated or total target proteins in lysed cells after stimulation are recognized by each primary antibody pair. The NanoBiT conjugated secondary antibodies then recognize their cognate primary antibodies, resulting in close proximity of the NanoBiT subunits to form a functional enzyme that generates bright luminescence. b Workflow of NanoBiT cell-based immunoassay. A homogeneous and simple “Add and Read” cell-based assay format.

Fig. 2. Optimization of the NanoBiT cell-based immunoassay with detection of IκBα phosphorylation or degradation.

a In all, 50,000 seeded MCF-7 cells were untreated or treated with TNFα (50 ng per ml, 10 min). IκBα phosphorylation was measured by NanoBiT cell-based immunoassay using the indicated antibody pairs at the indicated concentrations in the reaction after addition of antibodies. Fold changes (TNFα-Treated/Non-Treated) were calculated and represented in a heat map style. b In all, 50,000 seeded MCF-7 cells were pretreated with MG132 (20 µM, 30 min) and then untreated or treated with TNFα (50 ng per ml, 30 min). IκBα phosphorylation was measured with various antibody incubation times (5, 10, 30, 60, 90, and 120 min) before the Nano-Glo reagent was added. Fold changes (TNFα-Treated/Non-Treated) were calculated for each time point and are shown above each pair of bars. c Various number of MCF-7 cells (from 3500 to 63,000) were seeded and incubated at 37 °C in a 5% CO₂ overnight. The cells were pretreated with MG132 (20 µM, 1 h) and were untreated or treated with TNFα (10 ng per ml, 30 min) before IκBα phosphorylation was measured by NanoBiT cell-based immunoassay as described in Material and Methods section. d Various numbers of MCF-7 cells (from 3500 to 63,000) were seeded and incubated at 37 °C in a 5% CO₂ overnight. The cells were untreated or treated with TNFα (10 ng per ml, 30 min). Total IκBα level was measured by NanoBiT cell-based immunoassay as described in Material and Methods section. For b–d results are presented as means ± S.E.M (n = 3 technical replicates, the data are representative of two or more experiments).

Fig. 3. Validation of NanoBiT cell-based immunoassay detection of phosphorylated and total IκBα.

a, b In all, 50,000 seeded MCF-7 cells were untreated or pretreated with IKK16 (10 µM, 1 h) and then untreated or treated with TNFα (50 ng per ml, 10 min). Phosphorylated (a) or total IκBα (b) levels were measured by NanoBiT cell-based immunoassay using antibody pairs as described in Material and Methods. Results are presented as means ± S.E.M. (n = 3 technical replicates, the data are representative of two or more experiments). *P < 0.001 relative to Non-treated. c Various numbers of MCF-7 cells (from 3500 to 56,000) were seeded and incubated at 37 °C in a 5% CO₂ overnight. The cells were pretreated with MG132 (20 µM, 1 h) and then untreated or treated with TNFα (50 ng per ml, 30 min). IκBα phosphorylation was measured by NanoBiT cell-based immunoassay or Western blot analysis using the same primary antibodies. GAPDH blot served as a loading control. d Similar to c except cells were untreated or treated with only TNFα (50 ng per ml, 30 min). Total IκBα level was measured by NanoBiT cell-based immunoassay or western blot analysis using the same primary antibodies. GAPDH blot served as a loading control. Results for NanoBiT immunoassay in c, d are presented as means ± S.E.M. (n = 3 technical replicates, the data are representative of two or more experiments). Uncropped blots for c, d can be found in Supplementary Fig. 4.

Fig. 4. Deciphering NF-κB pathway activation by studying phosphorylation and degradation of IκBα.

In all, 50,000 seeded MCF-7 cells were untreated or treated with a TNFα (50 ng per ml) or b pretreated with MG132 (20 µM, 1 h) then TNFα (50 ng per ml) for various time points (2, 4, 6, 8, 10, 12, 15, and 30 min). Phosphorylated or total IκBα levels were measured by the corresponding NanoBiT cell-based immunoassay. c In all, 50,000 seeded MCF-7 cells were untreated or treated with TNFα (50 ng per ml) for a longer time course with Cycloheximide (20 µg per ml) added or not after the 30 min time point. Total IκBα level was measured by NanoBiT cell-based immunoassay. d, e Detection of phosphorylation and degradation of IκBα in human primary cells. d In all, 50,000 seeded human primary mammary epithelial cells or human breast tumor epithelial cells were pretreated with MG132 (20 µM, 1 h) before the cells were untreated or treated with TNFα (50 ng per ml) for various time points (2, 4, 6, 8, 10, 12, 15, 30, 45, and 60 min). Phosphorylation of IκBα was measured by the corresponding NanoBiT cell-based immunoassay. e, like d, except cells were treated or not with TNFα (50 ng per ml) in absence of MG132 for the various time points. Degradation of IκBα was measured by NanoBiT cell-based total IκBα immunoassay. Results are presented as means ± S.E.M. (n = 3 or four technical replicates, the data are representative of two or more experiments).

Fig. 5. Detection of total and phosphorylated target proteins in key signaling pathways using the NanoBiT cell-based immunoassay.

a, d In all, 50,000 seeded MCF-7 cells were left untreated or pretreated with IKK16 (10 µM, 1 h) and then were untreated or treated with TNFα (50 ng per ml, 10 min) before phosphorylated p65 (a) or total p65 (d) levels were measured. b, e 50,000 seeded MCF-7 cells were incubated at 37 °C in a 5% CO₂ overnight without serum. The cells were untreated or pretreated with LY294002 (20 µM, 1 h) and then were treated with insulin (1 µM, 10 min). Phosphorylated AKT (b) or total AKT (e) were measured by NanoBiT cell-based immunoassays. c, f 50,000 seeded A431 cells were incubated at 37 °C in a 5% CO₂ overnight without serum. The cells were untreated or pretreated with Ruxolitinib (1 µM, 1 h) and then were treated or not with IL-6 (10 ng per ml, 30 min) before phosphorylated STAT3 (c) or total STAT3 (f) were measured by NanoBiT cell-based immunoassays. Results are presented as means ± S.E.M. (n = 3 technical replicates, the data are representative of two or more experiments). *P < 0.001 relative to Non-treated.

Fig. 6. Effect of small and large molecules on phosphorylation or degradation of node proteins in key signaling pathways.

a In all, 50,000 seeded MCF-7 or human breast tumor epithelial cells were pretreated with various concentration of IKK16 for 1 h and then untreated or treated with TNFα (50 ng per ml, 30 min) before total IκBα was measured by NanoBiT cell-based immunoassays. b In all, 50,000 seeded MCF-7 cells were incubated at 37 °C in a 5% CO₂ overnight without serum. The cells were pretreated with various concentration of LY294002 for 1 h and then untreated or treated with insulin (1 µM, 10 min). Phosphorylated AKT was measured by NanoBiT p-AKT cell-based immunoassay as described. c–e 50,000 seeded A431 cells were incubated at 37 °C in a 5% CO₂ overnight without serum. The cells were pretreated with various concentration of Ruxolitinib or FM-381 (c), anti IL-6 antibody (d), or anti IL-6 receptor Tocilizumab (e) for 1 h and then were untreated or treated with IL-6 (10 ng per ml, 30 min). Phosphorylated STAT3 was measured by NanoBiT pSTAT3 cell-based immunoassay. Results are presented as means ± S.E.M. (n = 3 or four technical replicates, the data are representative of two or more experiments).

Fig. 7. Detection of natural and small molecule targeted degradation of proteins using NanoBiT cell-based immunoassay.

a In all, 50,000 seeded MCF-7 cells were treated with various concentration of TNFα for 30 min, before total IκBα level was measured. b In all, 50,000 seeded MCF-7 cells were incubated at 37 °C in a 5% CO₂ overnight without serum. The cells were treated with various concentration of Fulvestrant or Tamoxifen for 4 h. Total ER level was measured by NanoBiT cell-based immunoassay using ER primary antibodies described in Material and Methods. c In all, 50,000 seeded MCF-7 cells were incubated at 37 °C in a 5% CO₂ overnight without serum. The cells were untreated or treated with various concentration of Fulvestrant for 4 h. Total ER or IκBα level were measured by NanoBiT cell-based immunoassays. Results are presented as means ± S.E.M. (n = 3 technical replicates, the data are representative of two or more experiments).