Phospho-MLKL (Ser358) Antibody [K9E15]

Application: Reactivity: Human

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
54 kDa

Datasheet & SDS

Biological Description

Specificity
Phospho-MLKL (Ser358) Antibody [K9E15] detects endogenous levels of total MLKL protein only when it is phosphorylated at Ser358.

Clone
K9E15

Synonym(s)
FLJ34389, hMLKL, mixed lineage kinase domain like pseudokinase, mixed lineage kinase domain-like, Mixed lineage kinase domain-like protein, MLKL

Background
Phospho‑MLKL (Ser358) represents the activated executioner form of the mixed lineage kinase domain‑like pseudokinase within the necroptosis pathway, where MLKL acts downstream of RIPK3 to convert receptor‑proximal necrosome signaling into terminal membrane disruption and lytic cell death. The full‑length protein contains an N‑terminal four‑helix bundle executioner domain, a central brace region, and a C‑terminal pseudokinase domain whose activation loop harbors Thr357 and Ser358; this pseudokinase segment does not catalyze phosphate transfer but functions as a regulatory switch that senses phosphorylation by RIPK3 and allosterically releases the N‑terminal helical bundle to engage membranes. Necroptotic signaling initiated by death receptors, pattern‑recognition receptors, or other upstream inputs converges on RIPK3 activation and assembly of RIPK1–RIPK3–MLKL complexes, where RIPK3 phosphorylates the MLKL activation loop at Thr357 and Ser358, generating the phospho‑MLKL (Ser358) species that correlates with commitment to necroptotic execution. Phosphorylation at these residues drives a conformational transition in the pseudokinase domain that promotes dissociation of MLKL from RIPK3, exposure of the N‑terminal four‑helix bundle, and formation of higher‑order MLKL oligomers that translocate from the cytosol to the plasma membrane. Oligomerized, phospho‑MLKL inserts its N‑terminal helices into the inner leaflet of the plasma membrane and associates with specific phospholipids, where it perturbs membrane organization and integrity and forms disruptive assemblies that cause ion imbalance, swelling, and eventual membrane rupture characteristic of necroptotic cell death. Structural and biochemical analyses indicate that activation loop phosphorylation is sufficient to license MLKL killing activity, and phosphomimetic activation‑loop mutants bypass upstream signals to induce stimulus‑independent necrosis, underscoring the central regulatory position of the Thr357/Ser358 region and supporting the use of Ser358 phosphorylation as a precise molecular marker of MLKL activation state. Post‑translational modifications on MLKL beyond the activation loop, including additional phosphorylation events within the pseudokinase domain, further tune oligomerization efficiency, membrane engagement, and subcellular localization, placing Ser358 phosphorylation within a broader modification code that controls the amplitude and context of MLKL‑dependent necroptosis in infection and inflammatory settings. Phospho‑MLKL (Ser358) localizes predominantly to the plasma membrane and, in some contexts, to intracellular membranes at late stages of necroptosis, distinguishing it from nonphosphorylated MLKL and providing a spatial and biochemical readout of execution‑phase necroptotic signaling that is widely used to map necroptotic activity in tissues and disease models. In pathophysiological conditions, including ischemic injury, inflammatory diseases, and certain cancers, accumulation of phospho‑MLKL (Ser358) accompanies RIPK3 activation and necroptotic cell loss, linking this modification to tissue damage, inflammatory cytokine release, and modulation of the tumor microenvironment, while genetic or pharmacologic suppression of the RIPK3–MLKL axis reduces phospho‑MLKL formation and necroptotic output.

Background

Phospho‑MLKL (Ser358) represents the activated executioner form of the mixed lineage kinase domain‑like pseudokinase within the necroptosis pathway, where MLKL acts downstream of RIPK3 to convert receptor‑proximal necrosome signaling into terminal membrane disruption and lytic cell death. The full‑length protein contains an N‑terminal four‑helix bundle executioner domain, a central brace region, and a C‑terminal pseudokinase domain whose activation loop harbors Thr357 and Ser358; this pseudokinase segment does not catalyze phosphate transfer but functions as a regulatory switch that senses phosphorylation by RIPK3 and allosterically releases the N‑terminal helical bundle to engage membranes. Necroptotic signaling initiated by death receptors, pattern‑recognition receptors, or other upstream inputs converges on RIPK3 activation and assembly of RIPK1–RIPK3–MLKL complexes, where RIPK3 phosphorylates the MLKL activation loop at Thr357 and Ser358, generating the phospho‑MLKL (Ser358) species that correlates with commitment to necroptotic execution. Phosphorylation at these residues drives a conformational transition in the pseudokinase domain that promotes dissociation of MLKL from RIPK3, exposure of the N‑terminal four‑helix bundle, and formation of higher‑order MLKL oligomers that translocate from the cytosol to the plasma membrane. Oligomerized, phospho‑MLKL inserts its N‑terminal helices into the inner leaflet of the plasma membrane and associates with specific phospholipids, where it perturbs membrane organization and integrity and forms disruptive assemblies that cause ion imbalance, swelling, and eventual membrane rupture characteristic of necroptotic cell death. Structural and biochemical analyses indicate that activation loop phosphorylation is sufficient to license MLKL killing activity, and phosphomimetic activation‑loop mutants bypass upstream signals to induce stimulus‑independent necrosis, underscoring the central regulatory position of the Thr357/Ser358 region and supporting the use of Ser358 phosphorylation as a precise molecular marker of MLKL activation state. Post‑translational modifications on MLKL beyond the activation loop, including additional phosphorylation events within the pseudokinase domain, further tune oligomerization efficiency, membrane engagement, and subcellular localization, placing Ser358 phosphorylation within a broader modification code that controls the amplitude and context of MLKL‑dependent necroptosis in infection and inflammatory settings. Phospho‑MLKL (Ser358) localizes predominantly to the plasma membrane and, in some contexts, to intracellular membranes at late stages of necroptosis, distinguishing it from nonphosphorylated MLKL and providing a spatial and biochemical readout of execution‑phase necroptotic signaling that is widely used to map necroptotic activity in tissues and disease models. In pathophysiological conditions, including ischemic injury, inflammatory diseases, and certain cancers, accumulation of phospho‑MLKL (Ser358) accompanies RIPK3 activation and necroptotic cell loss, linking this modification to tissue damage, inflammatory cytokine release, and modulation of the tumor microenvironment, while genetic or pharmacologic suppression of the RIPK3–MLKL axis reduces phospho‑MLKL formation and necroptotic output.

References
https://pubmed.ncbi.nlm.nih.gov/33064829/ https://pubmed.ncbi.nlm.nih.gov/24703947/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%