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Protocol
Fluorometric Phospholipase Assays Based on Polymerized Liposome Substrates
Phospholipases are a diverse group of lipolytic enzymes with specificities for different sites on the phospholipid molecule (1). Based on the site of their hydrolytic attack, they can be classified into several g...
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Protocol
Efficient Immobilization of Phospholipase A2
Immobilized enzymes have been widely used for biomedical and industrial applications. Since phospholipase A2 (PLA2; E.C.3.1.1.4) catalyzes the hydrolysis of a wide range of phospholipid aggregates, immobilized PL...
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Protocol
Purification and Assay of Mammalian Group I and Group IIa Secretory Phospholipase A2
Phospholipases A2 (PLA2) are a family of ubiquitous lipolytic enzymes that are found both as intracellular and secreted proteins. Secretory PLA2s are small (14 kDa), homologous proteins that require millimolar Ca
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Article
Blockade of eosinophil migration and airway hyperresponsiveness by cPLA2-inhibition
We examined the role of a cytosolic phospholipase A2 (cPLA2) in antigen-induced eosinophil infiltration of airways and in airway hyperresponsiveness to methacholine. Inhibition of cPLA2, or blockade of the platel...
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Protocol
Bacterial Expression and Purification of C1 and C2 Domains of Protein Kinase C Isoforms
Protein kinase C (PKC) contains two types of membrane-targeting domains, C1 and C2 domains, in its regulatory region (1–5). It has been shown that C1 and C2 domains of conventional PKCs (α, βI, βII, and γ) and C1...
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Article
Characterization of calcium binding properties of lithostathine
The pancreas secretes primarily two types of metabolically important proteins: digestive enzymes and hormones. Lithostathine (LIT) is the only protein excreted from the pancreas that has no known digestive or...
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Article
The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain
Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishmen...
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Article
In situ quantitative imaging of cellular lipids using molecular sensors
Membrane lipids are dynamic molecules that play important roles in cell signalling and regulation, but an in situ imaging method for quantitatively tracking lipids in living cells is lacking at present. Here, we ...
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Article
Cholesterol modulates cell signaling and protein networking by specifically interacting with PDZ domain-containing scaffold proteins
Cholesterol is known to modulate the physical properties of cell membranes, but its direct involvement in cellular signaling has not been thoroughly investigated. Here we show that cholesterol specifically bin...
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Article
Cholesterol selectively activates canonical Wnt signalling over non-canonical Wnt signalling
Wnt proteins control diverse biological processes through β-catenin-dependent canonical signalling and β-catenin-independent non-canonical signalling. The mechanisms by which these signalling pathways are diff...
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Article
Open AccessAutophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate-specific phospholipase A1 activity
Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small int...
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Article
Open AccessErratum: Autophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate-specific phospholipase A1 activity
Nature Communications 6: Article number: 8745 (2015); Published: 26 October 2015; Updated: 27 November 2015 The original version of this Article contained an error in the spelling of the author Gilbert Di Paol...
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Protocol
High-Throughput Fluorometric Assay for Membrane–Protein Interaction
Membrane–protein interaction plays key roles in a wide variety of biological processes. To facilitate rapid and sensitive measurement of membrane binding of soluble proteins, we developed a fluorescence-based ...
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Article
Modeling Smith-Lemli-Opitz syndrome with induced pluripotent stem cells reveals a causal role for Wnt/β-catenin defects in neuronal cholesterol synthesis phenotypes
Analysis of SLOS patient-derived iPSCs reveals that cellular accumulation of the cholesterol precursor 7DHC, rather than cholesterol deficiency, causes dysregulation of Wnt/β-catenin signaling and aberrant neu...
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Article
Raft-based interactions of gangliosides with a GPI-anchored receptor
Monitoring new fluorescent ganglioside analogs at a single-molecule level suggests that gangliosides associate dynamically with GPI-anchored protein monomers, transient homodimer rafts, and clustered signaling...
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Article
Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol
Orthogonal cholesterol sensors that are useful for imaging cholesterol in the plasma membrane leaflets reveal an asymmetry in which a high outer leaflet concentration is important for signaling processes and i...
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Protocol
An In Situ Fluorescence Assay for Cholesterol Transporter Activity of the Patched
We recently developed a simultaneous in situ quantitative imaging technique for cholesterol in both leaflets of the plasma membrane of mammalian cells. This ratiometric fluorescence technique allows real-time ...
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Protocol
Lipid Binding of SH2 Domains
The Src homology 2 (SH2) domain is a modular protein interaction domain that specifically recognizes the phosphotyrosine (pY) motif of a target molecule. We recently reported that a large majority of human SH2...
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Article
Targeting lipid–protein interaction to treat Syk-mediated acute myeloid leukemia
Membrane lipids control the cellular activity of kinases containing the Src homology 2 (SH2) domain through direct lipid–SH2 domain interactions. Here we report development of new nonlipidic small molecule inh...
