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A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells.

Cell death and differentiation (2021-02-17)
André L Samson, Cheree Fitzgibbon, Komal M Patel, Joanne M Hildebrand, Lachlan W Whitehead, Joel S Rimes, Annette V Jacobsen, Christopher R Horne, Xavier J Gavin, Samuel N Young, Kelly L Rogers, Edwin D Hawkins, James M Murphy
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Lectin from Triticum vulgaris (wheat), biotin conjugate, lyophilized powder
Millipore
Benzonase® Nuclease, ≥250 units/μL, ≥90% (SDS-PAGE), recombinant, expressed in E. coli, buffered aqueous glycerol solution
Sigma-Aldrich
N-Ethylmaleimide, crystalline, ≥98% (HPLC)
Sigma-Aldrich
Donkey serum
Sigma-Aldrich
Anti-MLKL (58-70) antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5, clone 6C5, Chemicon®, from mouse
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Anti-hyaluronan synthase 1 Antibody, clone 12A2.3, clone 12A2.3, from mouse
Sigma-Aldrich
Anti-phospho-MLKL (Ser345) Antibody, clone 7C6.1, clone 7C6.1, from mouse
Sigma-Aldrich
Anti-MLKL Antibody, clone 3H1, clone 3H1, from rat