Studying Protein-Protein Interactions via Blot Overlay/Far Western Blot

Hall, Randy A.

doi:10.1007/978-1-4939-2425-7_24

Cited by 11 publications

(11 citation statements)

References 26 publications

(3 reference statements)

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“…Binding partners are detected from cell lysates through antibody interactions and enzymatic reactions [ 212 , 286 ]. Far Western Blot (biochemical): Proteins blotted to a membrane from an SDS-PAGE are probed for direct interaction with another protein that can be labeled or detected indirectly with specific antibodies [ 288 ]. Sandwich Screening Procedure (biochemical): Relies on protein–protein interactions to generate a specific DNA-binding activity.…”

Section: Appendix A1 In Vitro Interactionsmentioning

confidence: 99%

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Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix–Loop–Helix Transcription Factors

Torres-Machorro

2021

IJMS

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The basic helix–loop–helix transcription factor (bHLH TF) family is involved in tissue development, cell differentiation, and disease. These factors have transcriptionally positive, negative, and inactive functions by combining dimeric interactions among family members. The best known bHLH TFs are the E-protein homodimers and heterodimers with the tissue-specific TFs or ID proteins. These cooperative and dynamic interactions result in a complex transcriptional network that helps define the cell’s fate. Here, the reported dimeric interactions of 67 vertebrate bHLH TFs with other family members are summarized in tables, including specifications of the experimental techniques that defined the dimers. The compilation of these extensive data underscores homodimers of tissue-specific bHLH TFs as a central part of the bHLH regulatory network, with relevant positive and negative transcriptional regulatory roles. Furthermore, some sequence-specific TFs can also form transcriptionally inactive heterodimers with each other. The function, classification, and developmental role for all vertebrate bHLH TFs in four major classes are detailed.

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Section: Appendix A1 In Vitro Interactionsmentioning

confidence: 99%

“…Far Western Blot (biochemical): Proteins blotted to a membrane from an SDS-PAGE are probed for direct interaction with another protein that can be labeled or detected indirectly with specific antibodies [ 288 ].…”

Section: Appendix A1 In Vitro Interactionsmentioning

confidence: 99%

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix–Loop–Helix Transcription Factors

Torres-Machorro

2021

IJMS

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“…The interaction between L-FABP and VEGFR2 (intracellular domain) was evaluated using the overlay assay as previous described [ 49 ]. To estimate the affinity of L-FABP and VEGFR2, an increased amount of VEGFR2 purified protein (#P3871, Abnova, USA) was loaded on SDS-PAGE and transferred to a PVDF membrane.…”

Section: Methodsmentioning

confidence: 99%

Liver fatty acid-binding protein (L-FABP) promotes cellular angiogenesis and migration in hepatocellular carcinoma

Chung-Yu

Liu

et al. 2016

Oncotarget

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Liver fatty acid-binding protein (L-FABP) is abundant in hepatocytes and known to be involved in lipid metabolism. Overexpression of L-FABP has been reported in various cancers; however, its role in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated L-FABP and its association with vascular endothelial growth factors (VEGFs) in 90 HCC patients. We found that L-FABP was highly expressed in their HCC tissues, and that this expression was positively correlated with that of VEGF-A. Additionally, L-FABP significantly promoted tumor growth and metastasis in a xenograft mouse model. We also assessed the mechanisms of L-FABP activity in tumorigenesis; L-FABP was found to associate with VEGFR2 on membrane rafts and subsequently activate the Akt/mTOR/P70S6K/4EBP1 and Src/FAK/cdc42 pathways, which resulted in up-regulation of VEGF-A accompanied by an increase in both angiogenic potential and migration activity. Our results thus suggest that L-FABP could be a potential target for HCC chemotherapy.

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“…To detect enolase-plasminogen interactions, far-western blot assays were performed as described in Hall [61]. Briefly, the human plasminogen (Sigma-Aldrich, Saint Louis, MO, USA) (5 and 10 µg) and rNTHiENO (3 µg), obtained according to [20], were migrated in an SDS-PAGE (10%) and later electrotransferred to a nitrocellulose membrane (Bio-Rad, Inc, Hercules, CA, USA).…”

Section: Plasminogen-binding Assay By Far-western Blotmentioning

confidence: 99%

Structural Characterization of Haemophilus influenzae Enolase and Its Interaction with Human Plasminogen by In Silico and In Vitro Assays

et al. 2021

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Haemophilus influenzae is the causal agent of invasive pediatric diseases, such as meningitis, epiglottitis, pneumonia, septic arthritis, pericarditis, cellulitis, and bacteremia (serotype b). Non-typeable H. influenzae (NTHi) strains are associated with localized infections, such as otitis media, conjunctivitis, sinusitis, bronchitis, and pneumonia, and can cause invasive diseases, such as as meningitis and sepsis in immunocompromised hosts. Enolase is a multifunctional protein and can act as a receptor for plasminogen, promoting its activation to plasmin, which leads to the degradation of components of the extracellular matrix, favoring host tissue invasion. In this study, using molecular docking, three important residues involved in plasminogen interaction through the plasminogen-binding motif (251EFYNKENGMYE262) were identified in non-typeable H. influenzae enolase (NTHiENO). Interaction with the human plasminogen kringle domains is conformationally stable due to the formation of four hydrogen bonds corresponding to enoTYR253-plgGLU1 (K2), enoTYR253-plgGLY310 (K3), and enoLYS255-plgARG471/enoGLU251-plgLYS468 (K5). On the other hand, in vitro assays, such as ELISA and far-western blot, showed that NTHiENO is a plasminogen-binding protein. The inhibition of this interaction using polyclonal anti-NTHiENO antibodies was significant. With these results, we can propose that NTHiENO–plasminogen interaction could be one of the mechanisms used by H. influenzae to adhere to and invade host cells.

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Studying Protein-Protein Interactions via Blot Overlay/Far Western Blot

Cited by 11 publications

References 26 publications

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix–Loop–Helix Transcription Factors

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix–Loop–Helix Transcription Factors

Liver fatty acid-binding protein (L-FABP) promotes cellular angiogenesis and migration in hepatocellular carcinoma

Structural Characterization of Haemophilus influenzae Enolase and Its Interaction with Human Plasminogen by In Silico and In Vitro Assays

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