Proximity ligation assay

Fertilized eggs initiate translation of stored mRNAs in spatially and temporally controlled manners. Here, we present a protocol for visualizing spatial and temporal translation in zebrafish embryos by fluorescence in situ hybridization and proximity ligation assay. We describe steps for labeling newly synthesized proteins and mRNA, visualizing mRNA translation and mRNA, sample mounting, and observation. Coupling detection of mRNA molecules with their translation sites is useful for understanding the molecular and cellular mechanisms that drive embryo development...

Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat-attracting and insect-repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic, and evolutionary basis of iridoid biosynthesis in T...

The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome stalling and/or premature disassembly upon stress. Here, we characterize the Timeless-Tipin complex, a component of the fork protection complex. We used microscopy approaches, including colocalization analysis and proximity ligation assay, to investigate the spatial localization of the complex during ongoing replication in human cells. Taking advantage of the replication stress induction and the ensuing polymerase-helicase uncoupling, we characterized the Timeless-Tipin localization within the replisome...

While stroke represents one of the main causes of death worldwide, available effective drug treatment options remain limited to classic thrombolysis with recombinant tissue plasminogen activator (rtPA) for arterial-clot occlusion. Following stroke, multiple pathways become engaged in producing a vicious proinflammatory cycle through the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1) and heat shock protein 70 kDa (HSP72). HMGB1, in particular, can activate proinflammatory cytokine production when acetylated (AcHMGB1), a form that prefers cytosolic localization and extracellular release...

One of the hallmarks of Parkinson's disease (PD) is the alteration in the expression and function of NMDA receptor (NMDAR) and cannabinoid receptor 1 (CB1 R). The presence of CB1 R-NMDAR complexes has been described in neuronal primary cultures. The activation of CB1 R in CB1 R-NMDAR complexes was suggested to counteract the detrimental NMDAR overactivation in an AD mice model. Thus, we aimed to explore the role of this receptor complex in PD. By using Bioluminescence Resonance Energy Transfer (BRET) assay, it was demonstrated that α-synuclein induces a reorganization of the CB1 R-NMDAR complex in transfected HEK-293T cells...

BACKGROUND: Hypoxia is associated with poor prognosis in many cancers including glioblastoma (GBM). Glioma stem-like cells (GSCs) often reside in hypoxic regions and serve as reservoirs for disease progression. Long non-coding RNAs (lncRNAs) have been implicated in GBM. However, the lncRNAs that modulate GSC adaptations to hypoxia are poorly understood. Identification of these lncRNAs may provide new therapeutic strategies to target GSCs under hypoxia. METHODS: lncRNAs induced by hypoxia in GSCs were identified by RNAseq...

GGGGCC hexanucleotide repeat expansion in C9orf72 causes frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Expanded GGGGCC repeat RNA accumulates within RNA foci and is translated into toxic dipeptide repeat proteins; thus, efficient repeat RNA degradation may alleviate diseases. hnRNPA3, one of the repeat RNA-binding proteins, has been implicated in the destabilization of repeat RNA. Using APEX2-mediated proximity biotinylation, here, we demonstrate PABPC1, a cytoplasmic poly (A)-binding protein, interacts with hnRNPA3...

BACKGROUND: Chronic overconsumption of lipids followed by their excessive accumulation in the heart leads to cardiomyopathy. The cause of lipid-induced cardiomyopathy involves a pivotal role for the proton-pump vacuolar-type H+ -ATPase (v-ATPase), which acidifies endosomes, and for lipid-transporter CD36, which is stored in acidified endosomes. During lipid overexposure, an increased influx of lipids into cardiomyocytes is sensed by v-ATPase, which then disassembles, causing endosomal de-acidification and expulsion of stored CD36 from the endosomes toward the sarcolemma...

BACKGROUND: Although GqPCR activation often leads to cell survival by activating the PI3K/AKT pathway, it was previously shown that in several cell types AKT activity is reduced and leads to JNK activation and apoptosis. The mechanism of AKT inactivation in these cells involves an IGBP1-coupled PP2Ac switch that induces the dephosphorylation and inactivation of both PI3K and AKT. However, the machinery involved in the initiation of PP2A switch is not known. METHODS: We used phospho-mass spectrometry to identify the phosphorylation site of PP2Ac, and raised specific antibodies to follow the regulation of this phosphorylation...

BACKGROUND: As a key enzyme in ceramide synthesis, longevity assurance homologue 2 (LASS2) has been indicated to act as a tumour suppressor in a variety of cancers. Ferroptosis is involved in a variety of tumour processes; however, the role of LASS2 in regulating ferroptosis has yet to be explored. This article explores the potential underlying mechanisms involved. METHODS: Bioinformatics tools and immunohistochemical staining were used to evaluate LASS2 expression, and the results were analysed in relation to overall survival and clinical association in multiple cancers...

Nitric Oxide (NO) photocleavable donors are useful tools for interrogating nitric oxide signalling and have potential use in photopharmacological applications. There is currently intensive research into newer methods to improve NO release and kinetic profiles. Herein, we report the design and synthesis of a solid-supported photocleavable NO donor synthesized by ligating an N -nitroso photocleavable nitric oxide derivative to a TentaGel® polymer resin bead. Illumination with 365 nm light released nitric oxide that could be tracked via a turn-on fluorescence response (λex = 450 nm, λem = 545 nm) and measured using the Griess assay and diaminorhodamine derivatives...

Interorganelle contact sites regulate lipid metabolism, organelle dynamics and positioning, as well as apoptosis and autophagy. Here, we present a proximity ligation assay (PLA) protocol for measuring the association of two organelles in fixed cells. We describe steps for primary cell culture, primary cell transfection, and the assay itself. We then detail procedures for manual and image J-based analysis of PLA foci. This protocol optimizes the use of assay products and improves the identification of PLA foci labeling actual contact sites...

The identification of protein phosphatase 1 (PP1) holoenzyme substrates has proven to be a challenging task. PP1 can form different holoenzyme complexes with a variety of regulatory subunits, and many of those are cell cycle regulated. Although several methods have been used to identify PP1 substrates, their cell cycle specificity is still an unmet need. Here, we present a new strategy to investigate PP1 substrates throughout the cell cycle using clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 genome editing and generate cell lines with endogenously tagged PP1 regulatory subunit (regulatory interactor of protein phosphatase one, RIPPO)...

Rationale: Tripeptidyl peptidase II (TPP2) has been proven to be related to human immune and neurological diseases. It is generally considered as a cytosolic protein which forms the largest known protease complex in eukaryotic cells to operate mostly downstream of proteasomes for degradation of longer peptides. However, this canonical function of TPP2 cannot explain its role in a wide variety of biological and pathogenic processes. The mechanistic interrelationships and hierarchical order of these processes have yet to be clarified...

BACKGROUND: Pancreatic ductal adenocarcinoma is an aggressive cancer type with one of the lowest survival rates due to late diagnosis and the absence of effective treatments. A better understanding of PDAC biology will help researchers to discover the Achilles' heel of cancer cells. In that regard, our research team investigated the function of an emerging oncoprotein known as myoferlin. Myoferlin is overexpressed in PDAC and its silencing/targeting has been shown to affect cancer cell proliferation, migration, mitochondrial dynamics and metabolism...

Transcription-replication conflicts (TRCs) represent a potent endogenous source of replication stress. Besides the spatial and temporal coordination of replication and transcription programs, cells employ many additional mechanisms to resolve TRCs in a timely manner, thereby avoiding replication fork stalling and genomic instability. Proximity ligation assays (PLA) using antibodies against actively elongating RNA Polymerase II (RNAPIIpS2) and PCNA to detect proximity (<40nm) between transcribing RNA polymerases and replication forks can be used to assess and quantify TRC levels in cells...

Corepressors negatively regulate gene expression by chromatin compaction. Targeted regulation of gene expression could provide a means to control endothelial cell phenotype. We hypothesize that by targeting corepressor proteins, endothelial angiogenic function can be improved. To study this, the expression and function of nuclear corepressors in human umbilical vein endothelial cells (HUVEC) and in murine organ culture was studied. RNA-seq revealed that nuclear receptor corepressor 1 (NCoR1), silencing mediator of retinoid and thyroid hormone receptors (SMRT) and repressor element-1 silencing transcription factor (REST) are the highest expressed corepressors in HUVECs...

BACKGROUND: Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays an important role in diverse cellular processes by regulating Rho guanosine triphosphate (GTP)ases activity. RhoGDI1 phosphorylation regulates the spatiotemporal activation of Rho GTPases during cell migration. In this study, we identified polo-like kinase 1 (PLK1) as a novel kinase of RhoGDI1 and investigated the molecular mechanism by which the interaction between RhoGDI1 and PLK1 regulates cancer cell migration...

During the progression of pleural fibrosis, pleural mesothelial cells (PMCs) undergo a phenotype switching process known as mesothelial-mesenchymal transition (MesoMT). During MesoMT, transformed PMCs become myofibroblasts which produce increased extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1) that is critical to develop fibrosis. Here, we studied the mechanism that regulates FN1 expression in myofibroblasts derived from HPMCs. We found that myocardin (Myocd), a transcriptional coactivator of serum response factor (SRF) and a master regulator of smooth muscle and cardiac muscle differentiation, strongly controls HPMC FN1 gene expression...

A variety of commercial platforms are available for the simultaneous detection of multiple cytokines and associated proteins, often employing Ab pairs to capture and detect target proteins. In this study, we comprehensively evaluated the performance of three distinct platforms: the fluorescent bead-based Luminex assay, the proximity extension-based Olink assay, and a novel proximity ligation assay platform known as Alamar NULISAseq. These assessments were conducted on human serum samples from the National Institutes of Health IMPACC study, with a focus on three essential performance metrics: detectability, correlation, and differential expression...