Arige, Vikas Emrich, Scott M. Yoast, Ryan E. Trebak, Mohamed Yule, David I.
Published in
STAR Protocols
This protocol outlines steps to visualize and detect Ca2+ puffs following photo-liberation of caged inositol-1,4,5-trisphosphate (IP3) from HEK-293 cells expressing only the native IP3R type 1 receptor using total internal reflection fluorescence (TIRF) microscopy. TIRF microscopy offers high axial resolution and allows imaging at high speed, with ...
Lin, Yuan Watanabe-Chailland, Miki Zheng, Yi
Published in
STAR Protocols
Ras GTPases in complex with GTP or GTP analog exhibit dynamic equilibrium between two interconvertible conformations—an inactive state 1 and an active state 2. Unlike Ras, it remains unclear if the GTP-bound form of Rho GTPases also exhibits multiple conformational states. Here, we describe a protocol for structural and biochemical analyses of RhoA...
Clabbers, Max TB Holmes, Susannah Muusse, Timothy W Vajjhala, Parimala R Thygesen, Sara J Malde, Alpeshkumar K Hunter, Dominic JB Croll, Tristan Flueckiger, Leonie Nanson, Jeffrey D
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MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL(MAL TIR ) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88 TIR ). These crystals are too small for conventional X-ray cr...
Li, Na Hui, Hui Bray, Bill Gonzalez, Gwendolyn Michelle Zeller, Mark Anderson, Kristian G Knight, Rob Smith, Davey Wang, Yinsheng Carlin, Aaron F
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It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N6-methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of ...
Kankanamge, Dinesh Ubeysinghe, Sithurandi Tennakoon, Mithila Pantula, Priyanka Devi Mitra, Kishalay Giri, Lopamudra Karunarathne, Ajith
Published in
The Journal of biological chemistry
Phospholipase C β (PLCβ), which is activated by the Gq-family of heterotrimeric G proteins, hydrolyzes the inner membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), generating diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). Since Gq and PLCβ regulate many crucial cellular processes and have been identified as major disease driv...
Wang, Zhiyong Goto, Yusuke Allevato, Michael M Wu, Victoria H Saddawi-Konefka, Robert Gilardi, Mara Alvarado, Diego Yung, Bryan S O'Farrell, Aoife Molinolo, Alfredo A
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Immune checkpoint blockade (ICB) therapy has revolutionized head and neck squamous cell carcinoma (HNSCC) treatment, but
Manz, Kevin M. Siemann, Justin K. McMahon, Douglas G. Grueter, Brad A.
Published in
STAR Protocols
Patch-clamp and multi-electrode array electrophysiology techniques are used to measure dynamic functional properties of neurons. Whole-cell and cell-attached patch-clamp recordings in brain slices can be performed in voltage-clamp and current-clamp configuration to reveal cell-type-specific synaptic and cellular parameters governing neurotransmissi...
Clark, Iain C Gutiérrez-Vázquez, Cristina Wheeler, Michael A Li, Zhaorong Rothhammer, Veit Linnerbauer, Mathias Sanmarco, Liliana M Guo, Lydia Blain, Manon Zandee, Stephanie EJ
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Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon gui...
Haney, Meghan G. Wimsett, Mary Liu, Chunming Blackburn, Jessica S.
Published in
STAR Protocols
Dysregulation of Wnt signaling is a hallmark of many cancers, and the development of effective, non-toxic small-molecule Wnt inhibitors is desirable. Off-target toxicities of new compounds are typically tested in mouse models, which is both costly and time consuming. Here, we present a rapid and inexpensive protocol to determine the in vivo toxicit...
Martinez-Fabregas, Jonathan Pohler, Elizabeth Moraga, Ignacio
Published in
STAR Protocols
Stable isotope labeling by amino acid-based high-resolution phosphoproteomics is a powerful technique that allows for direct comparison of cells stimulated under different experimental conditions. This feature makes it the ideal methodology to identify cytokine signaling networks. Here, we present an optimized protocol for the isolation and identif...
