Abstract
Evaluating the steady-state protein level of the EGFR in live cells presents significant challenges compared to measuring its kinase activity. Traditional testing methods, such as immunoblotting, ELISA, and immunofluorescence assays, are generally restricted to fixed cells or cell lysates. Despite their utility, these methods are cumbersome and provide only intermittent snapshots of EGFR levels at specific time points. With emerging trends in drug development shifting toward engineering novel agents that promote protein degradation, rather than simply inhibiting kinase activity, a tool that enables real-time, quantitative detection of drug effects in live cells could catalyze advances in the field. Such an innovation would expedite the drug development process, enhancing the translation of research findings into effective, patient-centered therapies. The NanoLuc-EGFR cell line, created through CRISPR genome editing, allows for the continuous tracking and analysis of EGFR protein levels and their degradation within live cells. This approach provides quantitative monitoring of protein dynamics in real time, offering insights that go beyond absolute protein levels to include aspects such as protein stability and degradation rate. Using this cell line model, we observed that AT13387 and H84T BanLec induce EGFR degradation in A549-HiBiT cells, with the results confirmed by immunoblotting. In contrast, Erlotinib, Osimertinib, and Cetuximab inhibit EGFR phosphorylation without altering total EGFR levels, as validated by the HiBiT luciferase assay. The NanoLuc-EGFR cell line marks a significant advancement in understanding protein regulation and serves as an instrumental platform for investigating targeted therapies that modulate protein kinases, especially those that induce protein degradation.
Keywords: EGFR, Protein Degradation, HiBiT, Nano-Luciferase, Kinase Inhibitors
Introduction
The Epidermal Growth Factor Receptor (EGFR) plays a pivotal role in cellular processes, signaling growth and differentiation pathways upon binding with its ligand [1]. Over the past decades, EGFR has been implicated in various malignancies, mainly due to its over-expression or mutations. Consequently, the focus has predominantly been on understanding and therapeutically targeting its kinase activity [2]. However, EGFR possesses several functions independent of its kinase activity that are important in supporting cell survival by regulating glucose metabolism and response to stress. Additionally, EGFR has been linked to interactions with membrane proteins such as SLC7A11 (xCT), which is involved in amino acid transport and intracellular redox balance, and with G protein-coupled receptors (GPCRs), and integrins. These interactions collectively influence survival-promoting signaling pathways, which remain essential in various biological contexts, thus enhancing its significance in molecular biological landscapes [3–7].
Protein degradation constitutes a critical regulatory mechanism, pivotal to maintaining and modulating cellular homeostasis. Consequently, an emerging focus of oncology drug development has shifted towards engineering innovative agents that trigger the degradation of specific proteins, including EGFR. These agents harness the cell’s intrinsic machinery to promote protein degradation, offering a strategy that goes beyond merely inhibiting kinase activity [8,9].
Assessing EGFR degradation in live cells, however, poses significant challenges. Traditional methods such as immunoblotting, ELISA, or immunofluorescence assays are typically constrained to fixed cells or cell lysates, providing only intermittent snapshots of EGFR levels. Furthermore, these assays are cumbersome and do not efficiently capture the dynamic and complex process of intracellular protein degradation.
To address these challenges, recent advancements in bioluminescent tagging technologies have been utilized to incorporate an 11 amino acid peptide from luciferase, fusing it with the N-terminus of EGFR in A549 lung cancer cells via the CRISPR method. This technique allows for the monitoring of surface EGFR protein dynamics in real time, providing a tool for studying protein kinetics.
The development of the NanoLuc-EGFR cell line holds the potential to advance our understanding of EGFR and forms a promising tool for drug development. By allowing recurring quantification of EGFR protein levels in live cells, it provides valuable insights into protein trafficking, stability, and degradation. Furthermore, it can be used to optimize therapeutic agents capable of modulating EGFR degradation, fostering new advancements in oncology research and drug development. Thus, the establishment of the NanoLuc-EGFR cell line brings forth a cutting-edge technological landscape for informed cancer therapeutics.
Materials and Methods
Materials
AT13387 (Astex Pharmaceuticals, Cambridge, UK) was generously supplied by the CTEP, NIH, USA. Osimertinib (cat# HY-15772), and Erlotinib (cat# HY-12008) were procured from MedChem Express (Monmouth Junction, NJ). H84T BanLec was produced in E. coli as previously described [10]. (Erbitux (Cetuximab, C225) received from the University of Michigan’s pharmacy. EGFR (sc-03) was bought from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for phospho EGFR (cat# 2231), Hsp70 (cat# 4872), GAPDH (cat# 2118), pERK (cat# 4376), and total ERK (cat# 9107) were sourced from Cell Signaling Technology (Danvers, MA). Nano-Glo HiBiT extracellular detection system (cat# N2421), Nano-Glo Lytic detection system (cat# N3040), and CellTiter-Glo (cat# G7572) were procured from Promega Corporation (Madison, Wisconsin, USA). Phosphatase inhibitor (cat# P0001) and protease inhibitor cocktail (cat# P8340) were sourced from Millipore Sigma (Burlington, MA).
Cell Culture
The A549 lung cancer cells engineered to express NanoLuc-EGFR using the CRISPR/Cas9 approach were received as a gift from Promega Corporation (Madison, Wisconsin, USA). These cells are modified with the bioluminescent HiBiT tag, a small peptide derived from NanoLuc luciferase, enabling highly sensitive and quantitative tracking of protein dynamics in live cells. Both parental A549 (ATCC, Manassas, VA, USA) and HiBiT-expressing lines were cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum and 1x Penicillin-Streptomycin, ensuring consistent growth conditions for comparative analysis.
Optimization of Nano-Glo HiBiT Extracellular and Lytic Detection Reagents
1000 A549 parental and EGFR HiBiT cells per well were plated onto a 96-well plate and left to grow overnight. The following day, the media was replaced with FBS-free media, and cells were left to incubate for an hour. Both the Nano-Glo HiBiT extracellular and Lytic detection substrates were diluted in a buffer, and 50 μL of the diluted substrate was incubated with the cell culture for 10 minutes. The ensuing luminescence was recorded using Glo-Max Discover (Promega Corporation, Madison, Wisconsin, USA).
Optimization of A549-HiBiT EGFR Cell Density
A549 parental and EGFR HiBiT cells were plated onto a 96-well plate at densities ranging between 0 to 8000 cells for every well. Surface EGFR levels (extracellular detection) and total EGFR levels (Lytic detection) were quantified as stated above. Concurrently, total viable cell counts were determined using the Titer-Glo assay.
Immunoblotting
Cells were plated at a density of 0.6 million per 60 mm dish and incubated overnight to reach 70–80% confluence. Treatments were administered using either vehicle (DMSO) or varying concentrations of AT13387 (0–1000 nM), H84T BanLec (0–100 μg/mL), Cetuximab (0–300 μg/mL), Erlotinib (0–10 μM), or Osimertinib (0–1 μM). After a 24-hour incubation period, cells were harvested, and cell pellets were washed twice with ice-cold PBS and re-suspended in lysis buffer. This buffer consisted of 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% NP-40, 0.1% SDS, 1x phosphatase inhibitor (Thermo Scientific; Cat #PI78441), and 1x protease inhibitor cocktail (Sigma; Cat. No. P8340) for 30 minutes. Lysates were sonicated, and the particulate material was removed by centrifugation at 13,000 rpm for 10 minutes at 4°C. The clarified protein lysates were heated to 95°C for 5 minutes and subjected to SDS-PAGE on a 4%–12% Bis-Tris Precast Gel (Invitrogen), followed by transfer onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 hour at room temperature in blocking buffer containing 5% BSA and 1% normal goat serum in TBS [137 mM NaCl, 20 mM Tris-HCl (pH 7.6), 0.1% (v/v) Tween 20]. The primary antibody incubation occurred overnight at 4°C in the same buffer, followed by washing and 1-hour incubation with the horseradish peroxidase-conjugated secondary antibody. After three further washes in TBST, antibody binding was revealed using enhanced chemiluminescence plus reagent (Cytiva RPN2232). To quantify relative protein levels, immunoblot films were digitized and analyzed using ImageJ software (version 1.54ij). Relative protein levels shown are normalized to untreated control.
Immunofluorescence
Cells were plated onto sterile glass coverslips in 100-mm dishes. The following day, cells were treated with either vehicle (DMSO) or drugs. A day after treatments, coverslips were removed, and cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at room temperature, followed by a wash in PBS. Cells were then permeabilized in 0.5% Triton X-100 for 10 minutes and blocked in (5% goat serum and 1% BSA, in TBS) for an hour at room temperature. Cells were sequentially incubated with anti-phospho EGFR (cst #2231, 1:100) antibody or anti-EGFR (Santa Cruz Biotechnology, sc-03, 1:100) antibody overnight at 4°C. The slides were then washed with TBS thrice, incubated with the fluorescence-conjugated secondary antibodies for 1 hour (488 or 594 Alexa Fluor conjugated secondary antibody, 1:100 dilution), washed thrice, and fixed with a coverslip after ap-plying a drop of ProLong Gold anti-fade reagent with 4′, 6-diamidino-2-phenylindole (Molecular Probes) to each sample. Fluorescence images were captured using a Nikon Eclipse Ti2 microscope. The fluorescence intensity was quantified and normalized to the number of nuclei in the images. Ten random fields were reviewed, with at least 5 nuclei per field per treatment condition. The resultant values were then scaled from +1 (minimum intensity) to +3 (maximum intensity) and shown as semi-quantitative values.
Results
Development and Optimization of EGFR Detection Substrate Amount and Confirmation in HiBiT cells
To quantify EGFR protein expression with precision in live cells, the HiBiT system offers a highly sensitive bioluminescent assay based on complementation. This system involves a small peptide tag known as HiBiT, which specifically binds to a larger protein subunit called LgBiT. When the HiBiT tag, engineered into the target protein, in this case, EGFR, and LgBiT are brought into proximity, they form a functional luciferase enzyme complex [11]. This interaction occurs upon the addition of a substrate, which, upon catalysis by the luciferase complex, emits a bioluminescent signal directly proportional to the level of tagged protein present (Figure 1A). To assess EGFR protein levels in live cells accurately, optimization of several parameters was crucial, including the substrate concentration, the specificity of the bioluminescent signal, and the number of cells assayed for EGFR detection. Once conditions were refined, various compounds affecting EGFR protein levels were tested, with findings validated via immunoblotting and immunofluorescence.
Figure 1. Development and Optimization of EGFR Detection Substrate Amount and Confirmation in HiBiT cells.
(A) The schematic presents the split NanoLuc luciferase assay in A549 cells for detecting both surface and total EGFR expression. The 11 amino acid HiBiT tag, visualized as a blue triangle, is fused to the N-terminus of EGFR. Upon introduction of the complementary 156 amino acid LgBiT subunit, pre-conjugated with the luciferase substrate furimazine, the HiBiT and LgBiT subunits interact to reconstitute the active enzyme, which catalyzes a luminescent reaction. The presence of a lytic agent causes permeabilization of the cell membrane, depicted by a dotted plasma membrane, allowing LgBiT to access and bind with intracellular HiBiT-tagged EGFR. This results in bioluminescence that reflects total EGFR levels within the cells. Substrate optimization: One thousand A549 parental and HiBiT cells per well were plated in a 96-well plate and allowed to grow overnight. The following day, the media were replaced with FBS-free media, and the cells were incubated for 1 hour. The substrate was diluted in a buffer, and 50 μL of the diluted substrate was added to each well and incubated for 10 minutes. The resulting average surface luminescence (B) and the total luminescence (C) from at least three experiments were recorded using a GloMax Discover system and plotted along with the standard deviation.
Optimization of Substrate Amount
The initial recommended dilution of 1:100 for Nano-Glo HiBiT extracellular and lytic detection reagents (Promega, N2421 and N3030) was suboptimal for the A549-HiBiT EGFR cell line model. We refined the protocol to a 1:400 dilution for extracellular detection and 1:200 for lytic detection, which resulted in improved sensitivity and reduced background noise, thereby enhancing the overall signal-to-background ratio. Specificity trials with both parental A549 cells and those expressing HiBiT-tagged EGFR established that the luminescence was specific to the interaction of the HiBiT tag with LgBiT. No luminescent signal was detected in the parental cells, which affirmed the specificity of the assay (Figure 1B and C). These optimizations were crucial for the accurate quantification of EGFR levels.
Optimization of Cell Numbers
Following our adjustments to the Nano-Glo HiBiT detection reagents, we focused on determining the optimal number of cells for each well of a 96-well plate. It was established that a cell density of 1000 per well was sufficient for the effective detection of both surface-expressed and total EGFR (Figure 2A–B). We employed the CellTiter-Glo luminescent cell viability assay to quantify viable cells, contributing to the rigor of our findings (Figure 2C). In comparison with traditional immunoblotting, which required a minimum of 15,000 cells for reliable EGFR detection (Figure 2D), this optimized HiBiT protocol exhibited enhanced sensitivity. This could make it beneficial for experiments with limited cell numbers, such as high-throughput screening using 384-well plates, due to the assay’s sensitivity. Following the optimization of cell densities and reagent volumes, we utilized the Hsp90 inhibitor AT13387 and banana lectin H84T in the HiBiT assay system to validate the bioluminescence-based detection of EGFR protein reduction.
Figure 2. Optimization of Cell Numbers for EGFR Detection. (A-B) Cell Density Optimization:
Cells were plated in a 96-well plate at densities ranging from 0 to 8000 cells per well. Surface EGFR levels and total EGFR levels were quantified as described in Figure 1. (C) Concurrently, total viable cell counts were determined using the Titer-Glo assay., Results are presented as an average of at least three experiments +/− standard deviation (A, B, and C) and (D) 0.6 million A549-HiBiT EGFR-expressing cells were allowed to adhere and grow overnight. The next day, the cells were counted and aliquoted into 8 tubes with varying numbers (0 to 60,000) and were then processed for immunoblotting to detect EGFR levels. The EGFR band was detected in lysates prepared from 15,000 cells and above.
Validation of HiBiT Assay for EGFR Degradation Using AT13387
Building upon our previous research, in which we demonstrated that the inhibition of Hsp90 triggers the degradation of wild-type (WT)-EGFR [12] and induces compensatory upregulation of Hsp70 [13], we sought to validate these findings using the Hsp90 inhibitor, AT13387, in the A549-HiBiT EGFR cell line model. A549-HiBiT EGFR cells were treated with concentrations of AT13387 ranging from 0 to 1000 nM; a subsequent reduction in bioluminescence at 24 hours suggests degradation of EGFR (Figure 3A). The bioluminescence assay also demonstrated a time dependent decrease in EGFR levels at a 60 nM concentration of AT13387(Figure 3B). This correlation between the reduction in EGFR observed by immunoblotting (Figure 3C) and the decrease in bioluminescence validated the HiBiT assay as a method to detect the down regulation of EGFR, underscoring its utility for real-time analysis of surface EGFR dynamics in a cellular context.
Figure 3. Validation of EGFR degradation by AT13387.
(A) Dose-response relationship: 1000 A549-HiBiT cells were plated in a 96-well plate and treated with 0–1000 nM AT13387; luminescence was measured post 24-hour incubation. (B) Time-course analysis: 1000 A549-HiBiT cells were treated with 60 nM AT13387, and luminescence was measured periodically over 24 hours to assess the temporal effect of AT13387 on EGFR. (C) 0.6 million A549-HiBiT cells were seeded in a 60 mm dish and treated with AT13387 ranging from 0–1000 nM the following day. After 24 hours, cell lysates were collected for immunoblotting with specific antibodies. (D) Average EGFR levels of an immunoblotting data were quantified using ImageJ and plotted (error bars represent the SD from the average of three, separate experiments).
EGFR Degradation by H84T BanLec Treatment in A549 Cells Expressing HiBit-EGFR
We next assessed the HiBiT bioluminescence assay system’s capability to monitor the degradation of EGFR by the genetically engineered banana lectin H84T BanLec. While previous research has established H84T BanLec’s ability to induce EGFR degradation, our experiments leveraged the HiBiT-EGFR-expressing A549 as a model system to substantiate this assay’s effectiveness in tracking EGFR dynamics. Treatment with H84T BanLec resulted in a significant decrease in bioluminescence, indicative of reduced EGFR levels on the cell surface and within the whole-cell lysates, with the effect being concentration-dependent (Figure 4A). These bioluminescent observations were further validated by complementary techniques, including immunoblotting (Figure 4B) and immunofluorescence (Figure 6), which confirmed the reduction of EGFR protein following H84T BanLec exposure.
Figure 4. Validation of EGFR degradation by H84T BanLec.
(A) Dose-Response with H84T BanLec: One thousand A549-HiBiT cells were plated in a 96-well plate. The following day, cells were exposed to various concentrations of H84T BanLec (0–100 μg/ml). Readings were acquired at 24 hours. (B) Validation of EGFR degradation: 0.6 million A549-HiBiT cells were seeded in 60 mm dishes and treated with various concentrations of H84T BanLec (0–100 μg/ml). Cell lysates were prepared at 24 hours and subjected to immunoblotting using designated antibodies. (C) Average EGFR levels from the immunoblotting data were quantified using ImageJ and plotted; error bars represent the standard deviation (SD) from the average of three separate experiments.
Figure 6. Confirmation of EGFR Steady State and EGFR Phosphorylation Using Immunofluorescence.
A549-HiBiT-EGFR cells were seeded on coverslips in a 100 mm dish and treated with the following compounds: AT13387 (300 nM), H84T BanLec (100 μg/mL), Erlotinib (10 μM), Osimertinib (1 μM) or Cetuximab (300 μg/mL). The coverslips were collected after 24 hours of treatment. EGFR and phospho-EGFR were detected using anti-EGFR and anti-phospho-EGFR antibodies, respectively. DAPI was used to stain the nuclei. A semi-quantitative analysis (+, ++, +++) was performed through visual analysis of 50 cells in 3 random fields and shown below the corresponding panel (scale bars = 50 μm).
Effect of EGFR Tyrosine Kinase Inhibitors and Cetuximab on EGFR
Following observations of EGFR degradation with AT13387 and H84T BanLec, we aimed to further verify the model’s robustness using two EGFR tyrosine kinase inhibitors (Erlotinib and Osimertinib) and the anti-EGFR monoclonal antibody Cetuximab. Given the known mechanisms of EGFR tyrosine kinase inhibitors and Cetuximab, which primarily inhibit EGFR kinase activity without promoting its degradation, we hypothesized that these agents would not alter the HiBiT luciferase signal. As expected, treatment with Erlotinib, Osimertinib, and Cetuximab inhibited EGFR phosphorylation within 30 minutes (Supplementary Figure 2A and Figure 5, bottom panel). However, no significant changes were observed in bioluminescence (Figure 5, upper panel, and Supplementary Figures 2C–D), as total EGFR levels remained largely unaffected during this 24-hour time course analysis. These data confirm the suitability of this cell line model for monitoring EGFR steady-state levels and indicate its potential utility in screening for agents capable of inducing EGFR degradation.
Figure 5. Effects of Erlotinib, Osimertinib, and Cetuximab on Bioluminescence.
(upper panels). 1,000 A549-HiBiT cells were plated in a 96-well plate. The following day, the cells were exposed to various concentrations of Osimertinib (0–1 μM) (A), Erlotinib (0–10 μM) (B), or Cetuximab (0–300 μg/mL) (C). Bioluminescence from the surface or the entire cells was acquired at 24 hours. Data from three independent experiments are plotted. Validation of the Treatment Effect on EGFR and pEGFR (lower panels). 0.6 million A549-HiBiT cells were seeded in 60 mm dishes and treated with Osimertinib, Erlotinib, or Cetuximab as described above. Cell lysates were prepared at 24 hours and subjected to immunoblotting for pEGFR and EGFR. GAPDH was used as the loading control.
Confirmation of EGFR Modulation by Immunofluorescence Assay
To supplement bioluminescence and immunoblotting analyses, an immunofluorescence assay was employed to further confirm EGFR and pEGFR levels following treatment. A549-HiBiT cells were treated with AT13387 (300 nM), H84T BanLec (100 μg/ml), Erlotinib (10 μM), Osimertinib (1 μM), or Cetuximab (300 μg/ml) for 24 hours. Immunostaining utilized specific antibodies against EGFR and phospho-EGFR, differentiating total EGFR from its phosphorylated form, with cell nuclei visualized by DAPI staining. Immunofluorescence imaging confirmed the lack of significant change in total EGFR levels in cells treated with Erlotinib, Osimertinib, or Cetuximab. Conversely, reduced EGFR immunofluorescence was observed following AT13387 and H84T BanLec treatments, aligning with the diminished bioluminescence signals indicative of EGFR degradation (Figure 6). Phospho-EGFR staining distinguished between total and phosphorylated forms, verifying that bioluminescence changes were attributed to total EGFR reduction, not phosphorylation alterations, following treatment with H84T BanLec or AT13387. The effect of AT13387 and Osimertinib on EGFR and phospho-EGFR was also confirmed by immunofluorescence staining in HCC827 lung cancer cells (see Supplementary Figure 1). These findings further confirm that the A549-HiBiT cell model can effectively detect and quantitate EGFR protein expression.
Discussion
In this study, we have dissected the degradation kinetics of EGFR using a bioluminescent, NanoLuc luciferase-based assay. Our results confirm that using this system, both intracellular and cell surface EGFR can be measured with high sensitivity and specificity, and cell surface EGFR expression can be quantified in real-time. We observed that inhibition of Hsp90 or treatment with the lectin H84T BanLec led to a comparable rate of EGFR degradation in both membrane and whole cell contexts. Notably, in a cell line also expressing mutant KRAS, EGFR activity could be inhibited by the monoclonal antibody cetuximab and the tyrosine kinase inhibitors erlotinib and osimertinib without inducing degradation.
The specificity of the assay with no detected signal in A549 parental cells further underscores the reliability of this system in detecting the tagged EGFR. This level of precision positions the NanoLuc-EGFR assay as a valuable tool for high-throughput screenings to discover and evaluate modulators of EGFR expression and its associated signaling pathways.
The effectiveness of the cell line in detecting agents that promote EGFR degradation was demonstrated by the concentration-dependent responses to both the Hsp90 inhibitor, AT13387, and H84T BanLec. H84T BanLec, by binding specifically to high mannose glycans on EGFR, leads to its endocytosis and subsequent lysosomal degradation [14]. Furthermore, the system’s ability to differentiate between EGFR degradation and the inhibition of kinase activity by several inhibitors draws a clear distinction among the mechanisms that regulate EGFR signaling and turnover.
We presented data validating the EGFR-HiBiT tag system and anticipate that this system can be adapted for other biologically significant proteins to investigate protein trafficking, kinetics, protein-protein interactions, and can be used as a cell mode for drug library screening.
However, there exists the possibility that the HiBiT-tagged portion of EGFR may re-main stable despite the degradation of the protein, leading to a lingering bioluminescence signal from the intact HiBiT segment. Although the assay specificity has been validated, careful consideration is needed when interpreting luminescent data which could potentially reflect the persistence of degradation resistant HiBiT fragments instead of full-length EGFR protein.
To address this limitation, additional methods that could confirm the presence of full-length protein will be necessary, such as immunoprecipitation coupled with mass spectrometry or western blot analysis. These complementary techniques would provide a more comprehensive picture of EGFR degradation, ensuring more accurate validation of the luminescent measurements provided by the NanoLuc system.
The depth of our analysis reinforces the NanoLuc-EGFR A549 cell line as a sophisticated platform for elucidating EGFR regulation. By recognizing and navigating the limitations of the assay, our approach offers a productive avenue for advancing our under-standing of EGFR-targeted therapies, potentially impacting both drug development and fundamental biological research.
Conclusions
The development of the NanoLuc-EGFR cell line represents a significant step forward in the domain of EGFR research, providing an innovative methodology for the real-time observation of EGFR protein dynamics and degradation in living cells. The introduction of this cell line enhances the current arsenal of quantitative tools available for the study of EGFR, laying a foundation for advances in drug discovery processes that target this critical receptor. The implementation of the NanoLuc-EGFR cell line underscores the critical need for advanced cell-based assays capable of deciphering complex protein behavior. This technology promises to catalyze future research into protein degradation mechanisms, potentially accelerating the discovery and optimization of pharmaceutical compounds.
Supplementary Material
Acknowledgments:
Maureen Legendre produced H84T BanLec for these experiments, and the Promega Corporation provided the EGFR-HiBiT-expressing A549 cells as a gift.
Funding:
This research was funded by the NIH, with grants awarded to M.K.N. and T.S.L. under grant number R01CA248310. D.M.M. was supported by an NIH grant (R01AI175124), the Forbes Institute of the Rogel Cancer Center, and a Frankel Innovation Award at the University of Michigan. Maureen Legendre produced H84T BanLec for these experiments, and the Promega Corporation provided the EGFR-HiBiT-expressing A549 cells as a gift.
Footnotes
Conflict of interest: The authors declare no conflict of interest. The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interests, or non-financial interests, in the subject matter or materials discussed in this manuscript.
Ethics Statement: This study was conducted in accordance with the ethical standards of our institution. No human subjects or live animals were used in this research.
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