A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells
Details
Publication Year 2021-02-15, Volume 28, Issue #7, Page :2126-2144
Journal Title
Cell Death and Differentiation
Abstract
Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3, and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol- is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3, and MLKL signals.
Publisher
NPG
WEHI Research Division(s)
Inflammation; Advanced Technology And Biology
PubMed ID
33589776
NHMRC Grants
NHMRC/1124735 NHMRC/1124737
Rights Notice
Refer to copyright notice on published article.


Creation Date: 2021-03-09 01:37:06
Last Modified: 2021-08-17 10:36:48
An error has occurred. This application may no longer respond until reloaded. Reload 🗙