TY - JOUR
T1 - A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells
AU - Samson, André L.
AU - Fitzgibbon, Cheree
AU - Patel, Komal M.
AU - Hildebrand, Joanne M.
AU - Whitehead, Lachlan W.
AU - Rimes, Joel S.
AU - Jacobsen, Annette V.
AU - Horne, Christopher R.
AU - Gavin, Xavier J.
AU - Young, Samuel N.
AU - Rogers, Kelly L.
AU - Hawkins, Edwin D.
AU - Murphy, James M.
N1 - Funding Information:
Funding We are grateful to the Australian National Health and Medical Research Council for fellowship (JMH, 1142669; EDH, 1159488; JMM, 1172929), grant (1124735, 1124737, 1105023 and 2002965), and infrastructure (IRIISS 9000653) support, with additional support from
Publisher Copyright:
© 2021, The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.
PY - 2021/7
Y1 - 2021/7
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85101467821&partnerID=8YFLogxK
U2 - 10.1038/s41418-021-00742-x
DO - 10.1038/s41418-021-00742-x
M3 - Article
C2 - 33589776
AN - SCOPUS:85101467821
SN - 1350-9047
VL - 28
SP - 2126
EP - 2144
JO - Cell Death and Differentiation
JF - Cell Death and Differentiation
IS - 7
ER -