Search results for "Proximity ligation assay"
showing 10 items of 10 documents
Assessing the Histone Deacetylase Activity of SIRT6 in Primary Murine Hepatocytes via Proximity Ligation Assay
2016
Generation of primary cell culture of hepatocytes by mouse liver perfusion (MLP) combines the advantages of in vivo and in vitro models. It provides hepatocytes that grow under physiological conditions in mice, with the genotype of the whole organism or a specific gene knockout. In contrast to immortalized cell cultures, primary murine hepatocytes (pmHep) are non-cancerous cells with a limited lifespan but still amenable to classical in vitro methods such as treatment with drugs, small molecule inhibitors, and agonistic/antagonistic antibodies of surface receptors as well as transfection. One technique, which has gained popularity recently, is the analysis of protein-protein interactions by…
Detection, Analysis, and Quantification of GPCR Homo- and Heteroreceptor Complexes in Specific Neuronal Cell Populations Using the In Situ Proximity …
2018
GPCR’s receptosome operates via coordinated changes between the receptor expression, their modifications and interactions between each other. Perturbation in specific heteroreceptor complexes and/or their balance/equilibrium with other heteroreceptor complexes and corresponding homoreceptor complexes is considered to have a role in pathogenic mechanisms. Such mechanisms lead to mental and neurological diseases, including drug addiction, depression, Parkinson’s disease, and schizophrenia. To understand the associations of GPCRs and to unravel the global picture of their receptor–receptor interactions in the brain, different experimental detection techniques for receptor–receptor interactions…
Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer.
2016
Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca2+-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-1 were cleaved as assessed by western blot analysis. Both CAPNs were able …
The soluble form of pan-RTK inhibitor and tumor suppressor LRIG1 mediates downregulation of AXL through direct protein–protein interaction in gliobla…
2019
Abstract Background Targeted approaches for inhibiting epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs) in glioblastoma (GBM) have led to therapeutic resistance and little clinical benefit, raising the need for the development of alternative strategies. Endogenous LRIG1 (Leucine-rich Repeats and ImmunoGlobulin-like domains protein 1) is an RTK inhibitory protein required for stem cell maintenance, and we previously demonstrated the soluble ectodomain of LRIG1 (sLRIG1) to potently inhibit GBM growth in vitro and in vivo. Methods Here, we generated a recombinant protein of the ectodomain of LRIG1 (sLRIG1) and determined its activity in various cellular GBM mo…
Fibroblast Growth Factor Receptor 1– 5-Hydroxytryptamine 1A Heteroreceptor Complexes and Their Enhancement of Hippocampal Plasticity
2011
Background The hippocampus and its 5-hydroxytryptamine transmission plays an important role in depression related to its involvement in limbic circuit plasticity. Methods The analysis was made with bioluminescence resonance energy transfer, co-immunoprecipitation, in situ proximity ligation assay, binding assay, in cell western and the forced swim test. Results Using bioluminescence resonance energy transfer analysis, fibroblast growth factor receptor 1 (FGFR1)-5-hydroxytryptamine 1A (5-HT1A) receptor complexes have been demonstrated and their specificity and agonist modulation characterized. Their presence based on co-immunoprecipitation and proximity ligation assay has also been indicated…
XPO1E571K Mutation Modifies Exportin 1 Localisation and Interactome in B-cell Lymphoma
2020
The XPO1 gene encodes exportin 1 (XPO1) that controls the nuclear export of cargo proteins and RNAs. Almost 25% of primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) cases harboured a recurrent XPO1 point mutation (NM_003400, chr2:g61718472C>
Expression of p11 and Heteromeric TASK Channels in Rat Carotid Body Glomus Cells and Nerve Growth Factor–differentiated PC12 Cells
2020
Psychological stress activates the hypothalamus, augments the sympathetic nervous output, and elevates blood pressure via excitation of the ventral medullary cardiovascular regions. However, anatomical and functional connectivity from the hypothalamus to the ventral medullary cardiovascular regions has not been fully elucidated. We investigated this issue by tract-tracing and functional imaging in rats. Retrograde tracing revealed the rostral ventrolateral medulla was innervated by neurons in the ipsilateral dorsomedial hypothalamus (DMH). Anterograde tracing showed DMH neurons projected to the ventral medullary cardiovascular regions with axon terminals in contiguity with tyrosine hydroxyl…
Evidence for the existence of FGFR1-5-HT1A heteroreceptor complexes in the midbrain raphe 5-HT system.
2015
The ascending midbrain 5-HT neurons known to contain 5-HT1A autoreceptors may be dysregulated in depression due to a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by co-location of the FGFR1 and 5-HT1A immunoreactivities in midbrain raphe 5-HT cells, evidence for the existence of FGFR1–5-HT1A heteroreceptor complexes were obtained in the dorsal and median raphe nuclei of the Sprague–Dawley rat. Their existence in the rat medullary raphe RN33B cell cultures was also established. After combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA positive clusters was found in the RN33B cells. Similar results were reached upon coactivati…
In Situ Detection of Phosphorylated Platelet-derived Growth Factor Receptor β Using a Generalized Proximity Ligation Method
2007
Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a…
Structure-function analysis of peroxisomal ATP-binding cassette transporters using chimeric dimers
2014
Background: Peroxisomal ABC transporters are predicted to function as homodimers in mammals. [br/] Results: ABCD1 interacts with ABCD2. Chimeric proteins mimicking full-length dimers represent novel tools for functional study. Artificial homodimers and heterodimers are functional. [br/] Conclusion: Interchangeability between ABCD1 and ABCD2 is confirmed, but PUFA transport depends on ABCD2. [br/] Significance: For the first time, heterodimers in mammals are proven to be functional.[br/] ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 …