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EGF ligand stimulates EGFR-PDGFRA functional transactivation and heterodimerization in glioblastoma lines of varied genotype. Tumorsphere lines of indicated genotypes were serum-starved overnight followed by next-day treatment for 4 hours with the specified RTK-inhibitors. Cells were then ligand stimulated (as indicated) for 20 minutes. Whole cell lysate were collected and 30-mg of protein was run on SDS-PAGE and analyzed by western blot with the indicated antibodies. EGF stimulates PDGFRA phosphorylation, which is reversed by gefitinib and, to a lesser extent, PDGFB elicits detectable phosphorylation of EGFR in all lines: (a) TS753 – EGFR and PDGFRA focal co-amplification and (b) TS600 – EGFR focal amplification and Chromosome 7 gain (also refer to Supplementary Figure S2). (c, d) GTS-lines were serum starved overnight, treated for 4 hours with the indicated inhibitors and then stimulated for 20 minutes with 100ng/ml EGF. Cells were subsequently lysed and 2 mg of whole cell lysate were immunoprecipitated with total-EGFR antibody overnight. Beads were washed with lysis buffer, heat-denatured, run on SDS-PAGE and probed with activated and/or total PDGFRA antibodies in tumorsphere lines (c) TS753, and (d) TS600. EGF induced co-immunoprecipitation of EGFR with activated PDGFRA (pY720) in TS753, TS600. Coimmunoprecipitation between EGFR and PDGFRA was reversed by gefitinib and, for TS753, lapatinib as well. Reverse IP with PDGFRA antibody was less efficient but also demonstrated EGFR co-IP reversed by gefitinib (Supplementary Figure 3a). Coimmunoprecipitation between EGFR and activated PDGFRA (pY720) was also observed in EGFR-vIII positive TS12017 (Supplementary Figure 3b). (e) In situ Proximity Ligation Assay using proximity probes against EGFR and PDGFRA was performed in co-amplified tumor sphere line TS753. Eight-chamber slides seeded TS753 cells were serum starved overnight followed by 4-hour treatment with the indicated inhibitors. Cells were stimulated with EGF for 20-minutes. Cells were counter stained with DAPI (blue) to visualize the nucleus and red-dots show fluorophore expression due to proximity of oligo-tagged EGFR and PDGFRA antibodies associated with EGFR and PDGFRA interaction. Treatment of cells with gefitinib resulted in loss of the EGFR/PDGFRA interaction and thus a loss of in situ PLA signals (red dots), but treatment with imatinib did not.
Source publication
Concurrent amplifications of EGFR and PDGFRA have been reported in up to 5% of glioblastoma (GBM) and it remains unclear why such independent amplification events, and associated receptor overexpression, would be adaptive during glioma evolution. Here, we document that EGFR and PDGFRA protein co-expression occurs in 37% of GBM. There is wide cell-t...
Contexts in source publication
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... reported the unexpected de-phosphorylation of PDGFRA at the Y720 site by EGFR inhibitors gefitinib or lapatinib in an EGFR/PDGFRA co-amplified and co-expressing line TS753 9 . Here, we observed that EGF-ligand stimulated PDGFRA at Y720 in GTS-lines of different genotypes and this was reversed by EGFR inhibitors gefitinib, lapatinib and cetuximab (Fig. 2a,b and Supplementary Fig. ...
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... previously reported the unexpected de-phosphorylation of PDGFRA at the Y720 site by EGFR inhibitors gefitinib or lapatinib in an EGFR/PDGFRA co-amplified and co-expressing line TS753 9 . Here, we observed that EGF-ligand stimulated PDGFRA at Y720 in GTS-lines of different genotypes and this was reversed by EGFR inhibitors gefitinib, lapatinib and cetuximab (Fig. 2a,b and Supplementary Fig. S2). ...
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... 18 and PDGFRB 19 . Additionally PDGFRA has been shown to heterodimerize with FGFR1 20 . To investigate whether direct EGFR-PDGFRA receptor interaction may occur in glioblastoma cells we used co-immunopreciptation with/without EGF-stimulation to measure whether ligand-induced heterodimerization occurred in the co-amplified tumor sphere line TS753 (Fig. 2c), EGFR-amplified line TS600 ( Fig. 2d and Supplementary Fig. 3a) and EGFRvIII-expressing line TS12017 ( Supplementary Fig. S3b). Phosphorylated PDGFRA was pulled down with EGFR only in the condition of EGF stimulation in all GTS-lines tested, and this interaction was inhibited by EGFR inhibitor gefitinib (Fig. 2c,d). Consistent with ...
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... has been shown to heterodimerize with FGFR1 20 . To investigate whether direct EGFR-PDGFRA receptor interaction may occur in glioblastoma cells we used co-immunopreciptation with/without EGF-stimulation to measure whether ligand-induced heterodimerization occurred in the co-amplified tumor sphere line TS753 (Fig. 2c), EGFR-amplified line TS600 ( Fig. 2d and Supplementary Fig. 3a) and EGFRvIII-expressing line TS12017 ( Supplementary Fig. S3b). Phosphorylated PDGFRA was pulled down with EGFR only in the condition of EGF stimulation in all GTS-lines tested, and this interaction was inhibited by EGFR inhibitor gefitinib (Fig. 2c,d). Consistent with this data, receptor interaction was also ...
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... tumor sphere line TS753 (Fig. 2c), EGFR-amplified line TS600 ( Fig. 2d and Supplementary Fig. 3a) and EGFRvIII-expressing line TS12017 ( Supplementary Fig. S3b). Phosphorylated PDGFRA was pulled down with EGFR only in the condition of EGF stimulation in all GTS-lines tested, and this interaction was inhibited by EGFR inhibitor gefitinib (Fig. 2c,d). Consistent with this data, receptor interaction was also observed using proximity ligation assay 21 with/with- out EGF-stimulation (Fig. 2e). We found that EGFR-PDGFRA heterodimerization ( Tumorsphere lines of indicated genotypes were serum-starved overnight followed by next-day treatment for 4 hours with the specified ...
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... ( Supplementary Fig. S3b). Phosphorylated PDGFRA was pulled down with EGFR only in the condition of EGF stimulation in all GTS-lines tested, and this interaction was inhibited by EGFR inhibitor gefitinib (Fig. 2c,d). Consistent with this data, receptor interaction was also observed using proximity ligation assay 21 with/with- out EGF-stimulation (Fig. 2e). We found that EGFR-PDGFRA heterodimerization ( Tumorsphere lines of indicated genotypes were serum-starved overnight followed by next-day treatment for 4 hours with the specified RTK-inhibitors. Cells were then ligand stimulated (as indicated) for 20 minutes. Whole cell lysate were collected and 30 micrograms of protein was run on ...
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... cell lysate were collected and 30 micrograms of protein was run on SDS- PAGE and analyzed by western blot with the indicated antibodies. EGF stimulates PDGFRA phosphorylation, which is reversed by gefitinib and, to a lesser extent, PDGFB elicits detectable phosphorylation of EGFR in all lines: (a) TS753 -EGFR and PDGFRA focal co-amplification and (b) TS600 -EGFR focal amplification and Chromosome 7 gain (also refer to Supplementary Fig. S2). (c,d) GTS-lines were serum starved overnight, treated for 4 hours with the indicated inhibitors and then stimulated for 20 minutes with 100 ng/ml EGF. ...
Citations
... RTK will bind epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) [19][20][21]. EGFR and PDGFR are the two receptors with the most mutations expressed [22][23]. Decreased RTK activity will reduce EGFR and PDGFR. This decrease in EGFR and PDGFR levels is expected to reduce the angiogenesis process in GBM cells [24]. ...
The aim of this study was to determine differences in expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR)) in glioblastoma cell cultures in vitro when exposed to electric fields. This work followed a true experimental research design with a post-test only control group design. Glioblastoma cell culture was divided into 2 groups, namely the glioblastoma cell culture group exposed to electric field and those that were not exposed. This tool provided a voltage of 10, 30, and 50 peak to peak Voltage (Vpp) with a frequency of 100 KHz alternating current (AC). Evaluation of EGFR and PDGFR expressions was carried out after exposure. Exposure to a weak electric field with medium frequency for 24, 48, and 72 hours decreased the expression of EGFR and PDGFR in the angiogenesis process of glioblastoma cell cultures in vitro but not significantly. Further research is needed with a larger range of doses and treatment duration.
... One of the most critical factors determining anti-oncogenic protection and its nature is the NF1 gene, present in about 20% of GBM [12][13][14]. The platelet-derived growth factor receptor type A (PDGFRA) gene amplifies in 15% of GBM cases [15][16][17]. ...
Background:
Digital pathology has come a long way in terms of creating tools to improve existing diagnostic approaches. However, several pathology fields, such as neuropathology, are still characterized by low coverage from machine learning tools and neural network analysis, which may be due to the complexity of the internal cellular and molecular structure of the corresponding neoplasms, including glioblastomas.
Method:
In the framework of this study, using advanced proprietary tools for obtaining images of histological slides and their deep morphometric analysis, we studied samples of 198 patients with glioblastoma with the selection of morphometric cell clusters. Also, cells of each cluster were isolated, and their proliferative, migratory, invasive activity, survival ability, aerobic glycolysis activity, and chemo- and radioresistance were studied.
Results:
Four morphometric clusters were identified, including small-cell cluster, paracirculonuclear cluster, hypochromic cluster, and macronuclear cluster, which significantly differed in morphometric parameters and functional parameters. Hypochromic cluster cells demonstrated the highest proliferation activity; macronuclear cluster was the most active glucose consumer; paracirculonuclear cluster had the most prominent migratory and invasive activity and hypoxia resistance; small-cell cluster demonstrated predominantly average values of all parameters. Moreover, additional analysis revealed the presence of a separate subcluster of stem cell elements that correspond in their molecular properties to glioma stem cells and are present in all four clusters. It also turned out that several key molecular parameters of glioblastoma, such as mutational modifications in the EGFR, PDGFRA, and NF1 genes, along with the molecular GBM subtype, are significantly correlated with the identified cell clusters.
Conclusions:
Thus, the results represent an up-and-coming innovation in the practical field of digital pathology and fundamental questions of glioma carcinogenesis.
... Principles for combinatory regulation of BMP signaling through competitive receptor-ligand interactions has been elegantly demonstrated by Antebi and colleagues (Antebi et al. 2017). Adding to the complexity of signaling through CK growth factors, binding to their receptors may also induce heterodimerization to other growth factor receptors, described for heterodimerization between EGFR and PDGFRA (Chakravarty et al. 2017), PDGFRB (Saito et al. 2001), VEGFR2 (Paul et al. 2020) or FGFR2 (Ferguson et al. 2021). ...
CCN proteins play important functions during development, in repair mechanisms following tissue injury, as well as in pathophysiologic mechanisms of metastasis of cancer. CCNs are secreted proteins that have a multimodular structure and are categorized as matricellular proteins. Although the prevailing view is that CCN proteins regulate biologic processes by interacting with a wide array of other proteins in the microenvironment of the extracellular matrix, the molecular mechanisms of action of CCN proteins are still poorly understood. Not dissuading the current view, however, the recent appreciation that these proteins are signaling proteins in their own right and may even be considered preproproteins controlled by endopeptidases to release a C-terminal bioactive peptide has opened new avenues of research. Also, the recent resolution of the crystal structure of two of the domains of CCN3 have provided new knowledge with implications for the entire CCN family. These resolved structures in combination with structural predictions based upon the AlphaFold artificial intelligence tool provide means to shed new light on CCN functions in context of the notable literature in the field. CCN proteins have emerged as important therapeutic targets in several disease conditions, and clinical trials are currently ongoing. Thus, a review that critically discusses structure - function relationship of CCN proteins, in particular as it relates to interactions with other proteins in the extracellular milieu and on the cell surface, as well as to cell signaling activities of these proteins, is very timely.
Graphical abstract
Suggested mechanism for activation and inhibition of signaling by the CCN protein family (graphics generated with BioRender.com ).
... In this study, SUSD4 was found to interact with the tyrosine-kinase receptors EGFR and PDGFRα in triple negative breast cancer cell lines. The two receptors have been found to engage in heterodimers [29,30] and it could be that a heterotrimeric complex is formed together with SUSD4. Although the interaction between SUSD4 and PDGFRα persisted in CRISPR/Cas9-mediated EGFR knockout cells, thus showing that the interaction between SUSD4 and PDGFRα occurs independent of the EGFR, it does not negate the existence of a plausible heterotrimeric complex. ...
Background:
Sushi domain-containing protein 4 (SUSD4) is a recently discovered protein with unknown cellular functions. We previously revealed that SUSD4 can act as complement inhibitor and as a potential tumor suppressor.
Methods:
In a syngeneic mouse model of breast cancer, tumors expressing SUSD4 had a smaller volume compared with the corresponding mock control tumors. Additionally, data from three different expression databases and online analysis tools confirm that for breast cancer patients, high mRNA expression of SUSD4 in the tumor tissue correlates with a better prognosis. In vitro experiments utilized triple-negative breast cancer cell lines (BT-20 and MDA-MB-468) stably expressing SUSD4. Moreover, we established a cell line based on BT-20 in which the gene for EGFR was knocked out with the CRISPR-Cas9 method.
Results:
We discovered that the Epithelial Growth Factor Receptor (EGFR) interacts with SUSD4. Furthermore, triple-negative breast cancer cell lines stably expressing SUSD4 had higher autophagic flux. The initiation of autophagy required the expression of EGFR but not phosphorylation of the receptor. Expression of SUSD4 in the breast cancer cells led to activation of the tumor suppressor LKB1 and consequently to the activation of AMPKα1. Finally, autophagy was initiated after stimulation of the ULK1, Atg14 and Beclin-1 axis in SUSD4 expressing cells.
Conclusions:
In this study we provide novel insight into the molecular mechanism of action whereby SUSD4 acts as an EGFR inhibitor without affecting the phosphorylation of the receptor and may potentially influence the recycling of EGFR to the plasma membrane.
... In addition, active coexpression of EGFR and PDGFRA has been shown to occur in 37% of GBs. The presence of functional transactivation of PDGFRA by EGFR and EGF-induced heterodimerization of PDGFRA receptors, which are eliminated by EGFR inhibitors, have also been found [57]. ...
The dissertation is devoted to the study of intratumoral heterogeneity of glioblastomas at the cellular and molecular levels.
... These genes are up-regulated in tumors and down-regulated in periphery cells. It has been reported that co-expression of EGFR and PDGFRA is a driver event early in GBM [25,26]. The heat-shock genes HSPA1A and HSPA1B show down-regulation in tumor and up-regulation in the periphery, and act as marker genes for neuron clusters. ...
Glioblastoma multiforme (GBM) is the most common infiltrating lethal tumor of the brain. Tumor heterogeneity and the precise characterization of GBM remain challenging, and the disease-specific and effective biomarkers are not available at present. To understand GBM heterogeneity and the disease prognosis mechanism, we carried out a single-cell transcriptome data analysis of 3389 cells from four primary IDH-WT (isocitrate dehydrogenase wild type) glioblastoma patients and compared the characteristic features of the tumor and periphery cells. We observed that the marker gene expression profiles of different cell types and the copy number variations (CNVs) are heterogeneous in the GBM samples. Further, we have identified 94 differentially expressed genes (DEGs) between tumor and periphery cells. We constructed a tissue-specific co-expression network and protein–protein interaction network for the DEGs and identified several hub genes, including CX3CR1, GAPDH, FN1, PDGFRA, HTRA1, ANXA2 THBS1, GFAP, PTN, TNC, and VIM. The DEGs were significantly enriched with proliferation and migration pathways related to glioblastoma. Additionally, we were able to identify the differentiation state of microglia and changes in the transcriptome in the presence of glioblastoma that might support tumor growth. This study provides insights into GBM heterogeneity and suggests novel potential disease-specific biomarkers which could help to identify the therapeutic targets in GBM.
... Meanwhile, genetic alterations of components of the PDGFRα-PI3K-AKT signaling pathway occur in up to 70% of GBM [16]. Moreover, co-overexpression and co-activation of PDGF Rα with EGFR often occur in GBM tumors without amplification of either gene [17][18][19] but with a typical feature of high angiogenesis such as the most common EGFRvIII mutant-overexpressing GBM [20,21]. ...
Background
Glioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy. Overexpression and constitutive activation of PDGFs and PDGFRα are observed in most GBM; however, available inhibitors targeting isolated signaling pathways are minimally effective. Therefore, better understanding of crucial mechanisms underlying GBM is needed for developing more effective targeted therapies.
Methods
Target genes controlled by HIF1α in GBM were identified by analysis of TCGA database and by RNA-sequencing of GBM cells with HIF1α knockout by sgRNA-Cas9 method. Functional roles of HIF1α, PDGFs and PDGFRs were elucidated by loss- or gain-of-function assays or chemical inhibitors, and compared in response to oxygen tension. Pharmacological efficacy and gene expression in mice with intracranial xenografts of primary GBM were analyzed by bioluminescence imaging and immunofluorescence.
Results
HIF1α binds the PDGFD proximal promoter and PDGFRA intron enhancers in GBM cells under normoxia or mild-hypoxia to induce their expression and maintain constitutive activation of AKT signaling, which in turn increases HIF1α protein level and activity. Paradoxically, severe hypoxia abrogates PDGFRα expression despite enhancing HIF1α accumulation and corresponding PDGF-D expression. Knockout of HIF1A , PDGFD or PDGFRA in U251 cells inhibits cell growth and invasion in vitro and eradicates tumor growth in vivo. HIF1A knockdown in primary GBM extends survival of xenograft mice, whereas PDGFD overexpression in GL261 shortens survival. HIF1α inhibitor Echinomycin induces GBM cell apoptosis and effectively inhibits growth of GBM in vivo by simultaneously targeting HIF1α-PDGFD/PDGFRα-AKT feedforward pathway.
Conclusions
HIF1α orchestrates expression of PDGF-D and PDGFRα for constitutive activation of AKT pathway and is crucial for GBM malignancy. Therefore, therapies targeting HIF1α should provide an effective treatment for GBM.
... PDGFRA amplification is the more common alteration and occurs in 13.1% of GBM . While concurrent amplification of EGFR and PDGFRA has been reported in up to 5% of GBM , EGFR and PDGFRA coexpression at the mRNA level is seen in 37% of GBM sphere lines as reported by Chakravarty et al .The paper also showed functional transactivation of PDGFRα by EGFR: EGF stimulation in this setting can result both in EGFR-EGFR homodimerization as well as EGFR-PDGFRα heterodimerization which can drive proliferation(15). Further supporting this interaction, Hegi et al. observed that the expression of p-EGFR correlated with p-PDGFRβ in a window of opportunity study of 22 patients with recurrent GBM who were treated with at least 5 days of gefitinib (an EGFR inhibitor) prior to reresection . ...
... A c c e p t e d M a n u s c r i p t 15 A c c e p t e d M a n u s c r i p t 16 ...
Background
Glioblastoma remains a deadly brain cancer with dismal prognosis. Genetic alterations, including IDH mutations, 1p19q co-deletion status and MGMT promoter methylation have been proven to be prognostic and predictive to response to treatment in gliomas. In this manuscript, we aimed to correlate other mutations and genetic alterations with various clinical endpoints in patients with IDH-wild-type (IDHwt) glioblastoma.
Methods
We compiled a comprehensive clinically annotated database of IDHwt GBM patients treated at the Ohio State University Wexner Medical Center for whom we had mutational data through a CLIA-certified genomic laboratory. We then added data that is publicly available from Memorial Sloan Kettering Cancer Center through cBioPortal. Each of the genetic alterations (mutations, deletions and amplifications) served as a variable in univariate and multivariate Cox proportional hazard models.
Results
A total of 175 IDHwt GBM patients with available MGMT promoter methylation data from both cohorts were included in the analysis. As expected, MGMT promoter methylation was significantly associated with improved overall survival (OS). Median OS for MGMT promoter methylated and unmethylated GBM was 26.5 and 18 months, respectively (HR 0.45; p=0.003). Moreover, EGFR/ERBB alterations were associated with favorable outcome (HR of 0.37 (p=0.003), but only in MGMT promoter unmethylated GBM. We further found that patients with EGFR/ERBB alterations who also harbored PDGFRA amplification had a significantly worse outcome (HR 7.89; p=0.025).
Conclusions
Our data provides further insight into the impact of genetic alterations on various clinical outcomes in IDHwt GBM in two cohorts of patients with detailed clinical information and inspire new therapeutic strategies for IDHwt GBM.
... Two possibilities can be considered to explain it: simultaneous formation of two types of the complexes, -α7-nAChR/EGFR and α7-nAChR/PDGFR, both of which can bind SLURP-1, or formation of a complex between α7-nAChR and the EGFR/PDGFR heterodimer. Previously, the direct interaction of α7-nAChR with EGFR (Chernyavsky et al., 2015) and the possible formation of functional heterodimeric complexes EGFR/PDGFR (Saito et al., 2001;Perrone et al., 2009;Chakravarty et al., 2017) were reported. Thus, regardless of choice of the correct model, we have shown here for the first time that complexes of RTK and α7-nAChR can interact with a common modulator. ...
Secreted Ly6/uPAR-related protein 1 (SLURP-1) is a secreted Ly6/uPAR protein that negatively modulates the nicotinic acetylcholine receptor of α7 type (α7-nAChR), participating in control of cancer cell growth. Previously we showed, that a recombinant analogue of human SLURP-1 (rSLURP-1) diminishes the lung adenocarcinoma A549 cell proliferation and abolishes the nicotine-induced growth stimulation. Here, using multiplex immunoassay, we demonstrated a decrease in PTEN and mammalian target of rapamycin (mTOR) kinase phosphorylation in A549 cells upon the rSLURP-1 treatment pointing on down-regulation of the PI3K/AKT/mTOR signaling pathway. Decreased phosphorylation of the platelet-derived growth factor receptor type β (PDGFRβ) and arrest of the A549 cell cycle in the S and G2/M phases without apoptosis induction was also observed. Using a scratch migration assay, inhibition of A549 cell migration under the rSLURP-1 treatment was found. Affinity extraction demonstrated that rSLURP-1 in A549 cells forms a complex not only with α7-nAChR, but also with PDGFRα and epidermal growth factor receptor (EGFR), which are known to be involved in regulation of cancer cell growth and migration and are able to form a heterodimer. Knock-down of the genes encoding α7-nAChR, PDGFRα, and EGFR confirmed the involvement of these receptors in the anti-migration effect of SLURP-1. Thus, SLURP-1 can target the α7-nAChR complexes with PDGFRα and EGFR in the membrane of epithelial cells. Using chimeric proteins with grafted SLURP-1 loops we demonstrated that loop I is the principal active site responsible for the SLURP-1 interaction with α7-nAChR and its antiproliferative effect. Synthetic peptide mimicking the loop I cyclized by a disulfide bond inhibited ACh-evoked current at α7-nAChR, as well as A549 cell proliferation and migration. This synthetic peptide represents a promising prototype of new antitumor drug with the properties close to that of the native SLURP-1 protein.
... We describe a protocol for visualization of a protein-protein interaction that is induced by clustering of transmembrane proteins. Antibody-mediated cross-linking can be used for inducing clustering of transmembrane proteins instead of natural ligands (Ibuka et al., 2015;Toki et al., 2016;Chakravarty et al., 2017;Damiano et al., 2020). Interaction between transmembrane and intracellular proteins is easily visualized by a proximity ligation assay (PLA) without cell lysis and can be observed using a microscope. ...
This protocol visualizes dynamic interaction between a transmembrane protein and an intracellular protein induced by clusterization/oligomerization of the transmembrane protein. Association-dissociation of the intracellular region of the transmembrane protein with cytoplasmic protein(s) is detected by proximity ligation assay. Since a transmembrane protein often resists extraction, biochemical analysis of its dynamic interaction with cytoplasmic effectors is cumbersome. This protocol quantitatively visualizes protein-protein interaction occurring in the membrane periphery, providing a powerful tool to elucidate signal transduction across the membrane.
For complete details on the use and execution of this protocol, please refer to Ooki et al. (2019).
