Fig 1 - uploaded by Zhenyuan Tang
Content may be subject to copyright.
Schematic procedure of coimmunoprecipitation (co-IP) assay. Cells either endogenously or exogenously expressing the proteins of interest are prepared and treated as necessary (step 1). Cell lysate is then prepared with proper lysis buffer (step 2). Weak and transient interactions may require an additional stabilization step (see Note 4). Next, resin-immobilized antibodies are added into total cell lysate to capture protein of interest and its direct interacting partners (step 3). Finally, resin-antibody-protein complexes are precipitated and analyzed by immunoblotting or other protein analyzing methods such as mass spectrometry (steps 4 and 5)
Source publication
While there are various approaches available to analyze protein–protein interactions, coimmunoprecipitation (co-IP) remains one of the most classic and commonly used methods to discover novel protein interactions or to determine the physical association of proteins. The assay begins with the preparation of total cell or tissue lysate in an appropri...
Context in source publication
Context 1
... interactions but not protein-protein complex formation, while ionic detergents such as Chaps disrupt both (see Note 1). Proteinase inhibitors (and phosphatase inhibitors if phosphorylation regulates protein-protein complex formation of interest) should be added in the lysis buffer to prevent protein degradation (or complex formation) (Fig. 1). Next, protein of interest and its physical interacting partners are captured by incu- bating the lysate with the antibody that specifically target the pro- tein of interest (see Note 2). Selection of a specific antibody is another key factor in the IP/co-IP. IgG-binding Protein A/G con- jugated resin (e.g., agrose, sepharose, or ...
Citations
... protein signal was visualized using chemiluminescence ECL kit (Thermo Fisher Scientific). In addition, HADHA and MDM2 protein-protein interaction was analyzed by Co-IP and the experimental procedures followed the provided in the literature [18]. ...
Glioma represents a notoriously aggressive and malignant tumor that targets the central nervous system, with a poor prognosis for patients. In this research, we set out to examine the role of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA) in glioma, its clinical significance, as well as its potential biological mechanisms. In this study, we used immunohistochemistry staining to assess the expression level of HADHA in glioma tissues. We also evaluated the correlation between HADHA expression and patient survival using the Kaplan–Meier method. To determine the role of HADHA in glioma cells, we conducted loss-of-function assays in vitro and in vivo. Additionally, we utilized co-immunoprecipitation and protein stability assays to investigate the potential mechanisms involving HADHA, MDM2, and p53 in glioma. Our research findings indicate that gliomas exhibit high levels of HADHA. Clinically, high expression of HADHA suggests an increased risk of malignant tumors, recurrence, and reduced survival rates. Functionally, knocking down HADHA can lead to decreased proliferation, enhanced apoptosis, and inhibited migration of glioma cells. Mechanistically, HADHA accelerates MDM2-mediated p53 ubiquitination through interaction with MDM2. Consistently, MDM2 knockdown or overexpression of p53 can attenuate the promoting effect of HADHA overexpression on the malignant progression of glioma. We have discovered a novel role of HADHA in promoting MDM2-mediated p53 ubiquitination, which contributes to the progression of glioma. This finding provides a new perspective to understand the pathogenesis of glioma and offers a potential target for developing innovative therapeutic strategies.
... The co-immunoprecipitation assay was performed following a standard protocol 85 . Briefly, the plasmids (pCS2-HA-TANGO6, pCS2-FLAG-TANGO6, pCS2-HA-RPB2, pCS2-GFP-N, pCS2-GFP-Lumen and pCS2-GFP-C) and Lipo8000™ Transfection Reagent were transfected into HeLa or U251 cells. ...
Coat protein complex I (COPI) vesicles mediate the retrograde transfer of cargo between Golgi cisternae and from the Golgi to the endoplasmic reticulum (ER). However, their roles in the cell cycle and proliferation are unclear. This study shows that TANGO6 associates with COPI vesicles via two transmembrane domains. The TANGO6 N- and C-terminal cytoplasmic fragments capture RNA polymerase II subunit B (RPB) 2 in the cis-Golgi during the G1 phase. COPI-docked TANGO6 carries RPB2 to the ER and then to the nucleus. Functional disruption of TANGO6 hinders the nuclear entry of RPB2, which accumulates in the cytoplasm, causing cell cycle arrest in the G1 phase. The conditional depletion or overexpression of TANGO6 in mouse hematopoietic stem cells results in compromised or expanded hematopoiesis. Our study results demonstrate that COPI vesicle-associated TANGO6 plays a role in the regulation of cell cycle progression by directing the nuclear transfer of RPB2, making it a potential target for promoting or arresting cell expansion.
... This is concerning since experimental validation is still susceptible to false results due to various factors such as the transient nature of the PPI, ineffective antibodies for experiments such as coimmunoprecipitation, and protein types (i.e. transmembrane protein) [110]. While HP PPI databases serve as the resources for interacting protein pairs, available resources for validated non-interacting protein pairs such as the Negatome database [111] and Russell negative collection [112] do not contain inter-species PPI. ...
... We used the HA-tag at the C-terminus of the Msh2 protein to immunoprecipitate the interaction protein of Msh2 in T. thermophila, followed by a mass spectrometry analysis to identify the interaction proteins [46]. The Msh2-3HA protein and the protein physically bound to Msh2 were captured together with the HA antibody. ...
... The Msh2-3HA protein and the protein physically bound to Msh2 were captured together with the HA antibody. A series of washes were necessary to elute the unbound proteins [46]. The threshold value of iBAQ WT/iBAQ Msh2HA was 0.05 (Table S4). ...
Mismatch repair (MMR) is a conserved mechanism that is primarily responsible for the repair of DNA mismatches during DNA replication. Msh2 forms MutS heterodimer complexes that initiate the MMR in eukaryotes. The function of Msh2 is less clear under different chromatin structures. Tetrahymena thermophila contains a transcriptionally active macronucleus (MAC) and a transcriptionally silent micronucleus (MIC) in the same cytoplasm. Msh2 is localized in the MAC and MIC during vegetative growth. Msh2 is localized in the perinuclear region around the MIC and forms a spindle-like structure as the MIC divides. During the early conjugation stage, Msh2 is localized in the MIC and disappears from the parental MAC. Msh2 is localized in the new MAC and new MIC during the late conjugation stage. Msh2 also forms a spindle-like structure with a meiotic MIC and mitotic gametic nucleus. MSH2 knockdown inhibits the division of MAC and MIC during vegetative growth and affects cellular proliferation. MSH2 knockdown mutants are sensitive to cisplatin treatment. MSH2 knockdown also affects micronuclear meiosis and gametogenesis during sexual development. Furthermore, Msh2 interacts with MMR-dependent and MMR-independent factors. Therefore, Msh2 is necessary for macronuclear stability, as well as micronuclear mitosis and meiosis in Tetrahymena.
... Traditional methods such as co-immunoprecipitation [6] or pull down [7] allow for the identification of numerous potential interaction candidates from crude lysates in a short period of time and thus are regarded as relatively high-throughput and useful techniques to understand protein interactions. However, these approaches suffer from several intrinsic drawbacks, such as the disruption of all native subcellular microenvironments, the loss of potentially important transient or weak interactions during sample preparation, or the unexpected non-specific labeling. ...
Protein‐protein interactions (PPIs) play critical roles in almost all cellular signal transduction events. Characterization of PPIs without interfering with the functions of intact cells is very important for basic biology study and drug developments. However, the ability to profile PPIs especially those weak/transient interactions in their native states remains quite challenging. To this end, many endeavors are being made in developing new methods with high efficiency and strong operability. By coupling with advanced fluorescent microscopy and mass spectroscopy techniques, these strategies not only allow us to visualize the subcellular locations and monitor the functions of protein of interest (POI) in real time, but also enable the profiling and identification of potential unknown interacting partners in high‐throughput manner, which greatly facilitates the elucidation of molecular mechanisms underlying numerous pathophysiological processes. In this review, we will summarize the typical methods for PPIs identification in living cells and their principles, advantages and limitations will also be discussed in detail.
... After that, protein signal was visualized through chemiluminescence ECL kit (Thermo Fisher Scientific) and GAPDH as load control. ZNF655 and CDK1 protein-protein interaction was analyzed by Co-IP assay and the experimental procedures were performed as previously described [25]. ...
Pancreatic cancer has an extremely terrible prognosis and is a common cause of cancer death. In this study, the clinic value, biological function and underlying mechanisms of Zinc finger protein 655 (ZNF655) in human pancreatic cancer were evaluated. The expression level of ZNF655 in pancreatic cancer was determined by immunohistochemistry (IHC) staining. The biological effects of ZNF655 in pancreatic cancer cells was investigated by loss/gain-of-function assays in vitro and in vivo. The downstream molecular mechanism of ZNF655 was explored using co-immunoprecipitation (Co-IP), dual-luciferase reporter and chromatin immunoprecipitation (Ch-IP). ZNF655 expression was significantly elevated in human pancreatic cancer and possessed clinical value in predicting poor prognosis. Functionally, ZNF655 knockdown inhibited the biological progression of pancreatic cancer cells, which was characterized by weaken proliferation, enhanced apoptosis, arrested cell cycle in G2, impeded migration, and suppressed tumor growth. Mechanistically, ZNF655 played an important role in promoting the binding of E2F transcription factor 1 (E2F1) to the cyclin-dependent kinase 1 (CDK1) promoter. Furthermore, knockdown of CDK1 alleviated the promoting effects of ZNF655 overexpression in pancreatic cancer cells. The promotive role of ZNF655 in pancreatic cancer via CDK1 was determined, which drew further interest regarding its clinical application as a promising therapeutic target.
... The co-immunoprecipitation (Co-IP) experiment was performed as previously described [24]. Briefly, protein lysates were prepared from rat LV myocardia of Sham (n = 3) and pressure overload (PO)-induced HFrEF (n = 3) groups, as well as from a tissue bank of human HFrEF LV myocardia (n = 3) obtained from patients at the time of LVAD implantation. ...
Previous work showed a role of BNIP3 in myocardial remodeling and progression to HFrEF. We utilized a multiomics approach to unravel BNIP3-related molecular mechanisms in the pathogenesis of HFrEF. BNIP3 knockdown in HFrEF improved glycolysis, pyruvate metabolism, branched-chain amino acid catabolism, and oxidative phosphorylation, and restored endoplasmic reticulum (ER)–mitochondrial (mt) calcium and ion homeostasis. These effects of BNIP3 on cardiac metabolism were related to its interaction and downregulation, and/or phosphorylation, of specific mt-proteins involved in the aforementioned metabolic pathways, including the MICOS and SLC25A families of carrier proteins. BNIP3 affected ER–mt-calcium and ion homeostasis via its interaction-induced VDAC1 dimerization and modulation of VDAC1 phosphorylation at Ser104 and Ser241, and the downregulation of LETM1. At the ER level, BNIP3 interacted with the enzyme SERCA2a and the PKA signaling complex, leading to the downregulation of SERCA2a and PKA-mediated Ser16 phospholamban phosphorylation. Additionally, BNIP3 attenuated AMPK and PRKCE activity by modulating AMPK phosphorylation at Ser485/491 and Ser377 residues, and PRKCE phosphorylation at Thr521 and Thr710 residues. BNIP3 also interacted with sarcomeric, cytoskeletal, and cellular transcription and translation proteins, and affected their expression and/or phosphorylation. In conclusion, BNIP3 modulates multiple pathobiological processes and constitutes an attractive therapeutic target in HFrEF.
... Another concern is that co-IP is not well suited to detect weak or transient interactions as the experimental procedure includes several washing steps, often with detergents, to eliminate nonspecific binding. Also, detection of proteins via western blot requires sufficient protein expression, which can be problematic if endogenous promoters are used, which are often only weakly or transiently expressed, or only expressed in a few cells (as reviewed in Tang and Takahashi, 2018). Another major drawback of this technique is that spatial information of the POI is lost through lysis of the cells. ...
Molecular processes depend on the concerted and dynamic interactions of proteins, either by one-on-one interactions of the same or different proteins or by the assembly of larger protein complexes consisting of many different proteins. Here, not only the protein-protein interaction (PPI) itself, but also the localization and activity of the protein of interest (POI) within the cell is essential. Therefore, in all cell biological experiments, preserving the spatio-temporal state of one POI relative to another is key to understand the underlying complex and dynamic regulatory mechanisms in vivo. In this review we examine some of the applicable techniques to measure PPI in planta as well as recent combinatorial advances of PPI methods to measure the formation of higher order complexes with an emphasis on in vivo imaging techniques. We compare the different methods and discuss their benefits and potential pitfalls to facilitate the selection of appropriate techniques by giving a comprehensive overview about how to measure in vivo PPI in plants.
... Many experimental biology methods have been devised to find pairs of interacting proteins. Benchwork experiments like co-immunoprecipitation (co-IP) are resource-intensive and low-throughput, often leading to false negatives depending on experimental conditions, for example due to ineffective antibodies or due to the transient nature of PPIs [1]. In contrast, high throughput screens such as yeast two-hybrid (Y2H) and tandem affinity purification mass-spectrometry (TAP-MS), and more recently a sequencing approach called PROPER-seq [2] attempt to capture tens-to-hundreds of thousands of PPIs in a single experiment. ...
Protein–protein interactions (PPIs) perform various functions and regulate processes throughout cells. Knowledge of the full network of PPIs is vital to biomedical research, but most of the PPIs are still unknown. As it is infeasible to discover all of them experimentally due to technical and resource limitations, computational prediction of PPIs is essential and accurately assessing the performance of algorithms is required before further application or translation. However, many published methods compose their evaluation datasets incorrectly, using a higher proportion of positive class data than occuring naturally, leading to exaggerated performance. We re-implemented various published algorithms and evaluated them on datasets with realistic data compositions and found that their performance is overstated in original publications; with several methods outperformed by our control models built on ‘illogical’ and random number features. We conclude that these methods are influenced by an over-characterization of some proteins in the literature and due to scale-free nature of PPI network and that they fail when tested on all possible protein pairs. Additionally, we found that sequence-only-based algorithms performed worse than those that employ functional and expression features. We present a benchmark evaluation of many published algorithms for PPI prediction. The source code of our implementations and the benchmark datasets created here are made available in open source.
... Co-IP was performed as previously described (Tang and Takahashi, 2018). 300 mg of protein from cells, protease inhibitor cocktail (Sigma-Aldrich, Shanghai, China), lysis buffer (Cell Signaling Technology, Beverly, USA), Protein A/G Magnetic bead slurry (Thermos Fisher Scientific Rockford, USA), 1 μg mouse anti-ATG4B antibody (Abcam), 1 μg rabbit anti-LC3 antibody (Abcam) and mouse IgG (Cell Signaling Technology) were used. ...
Head and neck squamous cell carcinoma is the sixth most common tumor worldwide, and half of head and neck squamous cell carcinoma patients are with oral squamous cell carcinoma (OSCC). 300,000 new cases of OSCC were reported annually. Even with multi-modality treatment, the prognosis of OSCC remains unsatisfactory. Thus, it is urgent to discover novel therapeutic targets for OSCC. Some microarray studies have revealed that Keratin 4 (KRT4) is downregulated in OSCC, whereas its role in OSCC development remains unknown. The present study revealed that KRT4 suppressed OSCC progression by inducing cell apoptosis and inhibiting cell invasion. In addition, KRT4 over-expression inhibited autophagy by blocking the interaction of autophagy-related 4B cysteine peptidase (ATG4B) and microtubule-associated protein 1A/1B light chain 3 (LC3) to regulate apoptosis and invasion of OSCC. In conclusion, KRT4 played an important role in OSCC development through regulating ATG4B-mediated autophagy and may be a novel therapeutic drug target of OSCC.
