Biology subjects

Vue, N.

Publications and source records attributed to Vue, N..

3 recordsLinked to original sources

bioRxiv2023

Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells

Proximity ligation assays (PLA) use specific antibodies to detect endogenous protein-protein interactions. PLA is a highly useful biochemical technique that allows two proteins within close proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of PLA in mouse skeletal muscle (SkM) is novel. In this article, we discuss how the PLA method can be used in SkM to study the protein-protein interactions within mitochondria-endoplasmic reticulum contact sites (MERCs). Tweetable AbstractProximity Ligation Assays can be used in skeletal muscle tissue and myoblasts to explore the protein-protein interactions involved in MERC sites. HighlightsO_LISkeletal muscle tissue and cells are plated on glass coverslips for evaluation by proximity ligation assay (PLA). C_LIO_LIFollowing fixation, cells are probed and stained for Mfn1, Mfn2, mitochondria, and ER and imaged using fluorescence confocal microscopy. C_LIO_LIThis method shows that PLA can be used in mouse SkM and is adaptable to other models. C_LIO_LIProtocol for detection of protein-protein interactions using PLA. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/541599v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1021ce7org.highwire.dtl.DTLVardef@1b78f8borg.highwire.dtl.DTLVardef@31030corg.highwire.dtl.DTLVardef@1bf0afa_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@169ba8corg.highwire.dtl.DTLVardef@566b30org.highwire.dtl.DTLVardef@197330dorg.highwire.dtl.DTLVardef@198ca7borg.highwire.dtl.DTLVardef@158b80_HPS_FORMAT_FIGEXP M_TBL C_TBL

cell biology↗

bioRxiv2023

Components of Isolated Skeletal Muscle Differentiated Through Antibody Validation

Isolation of skeletal muscles allows for the exploration of many complex diseases. Fibroblasts and myoblast play important roles in skeletal muscle morphology and function. However, skeletal muscles are complex and made up of many cellular populations and validation of these populations is highly important. Therefore, in this article, we discuss a comprehensive method to isolate mice skeletal muscle, create satellite cells for tissue culture, and use immunofluorescence to validate our approach. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/541600v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@165b34dorg.highwire.dtl.DTLVardef@1de9976org.highwire.dtl.DTLVardef@12d3cfeorg.highwire.dtl.DTLVardef@ff660c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Creating Optimal Conditions for OPA1 isoforms by Western Blot in Muscle Tissue

OPA1 is a dynamin-related GTPase that modulates various mitochondrial functions and is involved in mitochondrial morphology. There are eight different isoforms of OPA1 in humans and five different isoforms in mice that are expressed as short or long-form isoforms. These isoforms contribute to OPA1s ability to control mitochondrial functions. However, isolating OPA1 all long and short isoforms through western blot has been a difficult task. To address this issue, we outline an optimized western blot protocol to isolate 5 different isoforms of OPA1 on the basis of different antibodies. This protocol can be used to study changes in mitochondrial structure and function. Tweetable AbstractWestern blot protocol optimization to visualize OPA1 isoforms. HighlightsO_LIProtocol for isolating OPA1 isoforms in primary skeletal muscle myoblast and myotubes C_LIO_LISteps for running isolated skeletal muscle cells from muscle tissue on a gel C_LIO_LIHow to collect samples in preparation for western blotting C_LIO_LIDetection of OPA1 isoforms C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/541601v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1dc254dorg.highwire.dtl.DTLVardef@150d3eorg.highwire.dtl.DTLVardef@186ed2org.highwire.dtl.DTLVardef@13cc3a_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@1c518ceorg.highwire.dtl.DTLVardef@df826borg.highwire.dtl.DTLVardef@1eb5bf8org.highwire.dtl.DTLVardef@e324e6org.highwire.dtl.DTLVardef@8baedc_HPS_FORMAT_FIGEXP M_TBL C_TBL Method SummarySamples for western blot analysis are isolated from lysed cells, loaded onto a gel, and ran using optimized conditions to better isolate OPA1 isoforms. Samples are transferred to a membrane for incubation and protein detection using OPA1 antibodies.

cell biology↗