MLKL Antibody - #DF7412

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Product: MLKL Antibody
Catalog: DF7412
Description: Rabbit polyclonal antibody to MLKL
Application: WB IHC IF/ICC
Reactivity: Human, Mouse, Rat
Mol.Wt.: 54kDa; 54kD(Calculated).
Uniprot: Q8NB16
RRID: AB_2839350

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MSDS
Data Sheet
COA
Protocols
WB Handbook
IHC Protocol
IF/ICC Protocol

Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IHC 1:50-1:200, IF/ICC 1:100-1:500
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Mouse,Rat
Clonality:
Polyclonal
Specificity:
MLKL Antibody detects endogenous levels of total MLKL.
RRID:
AB_2839350
Cite Format: Affinity Biosciences Cat# DF7412, RRID:AB_2839350.
Conjugate:
Unconjugated.
Purification:
The antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin (Thermo Fisher Scientific).
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

9130019I15Rik; FLJ34389; hMLKL; Mixed lineage kinase domain like; Mixed lineage kinase domain like protein; Mixed lineage kinase domain like pseudokinase; Mixed lineage kinase domain-like protein; Mlkl; MLKL_HUMAN;

Immunogens

Immunogen:
Uniprot:

Q8NB16(MLKL_HUMAN) >>Visit HPA database.

Gene(ID):
MLKL(197259)
Sequence:
MENLKHIITLGQVIHKRCEEMKYCKKQCRRLGHRVLGLIKPLEMLQDQGKRSVPSEKLTTAMNRFKAALEEANGEIEKFSNRSNICRFLTASQDKILFKDVNRKLSDVWKELSLLLQVEQRMPVSPISQGASWAQEDQQDADEDRRAFQMLRRDNEKIEASLRRLEINMKEIKETLRQYLPPKCMQEIPQEQIKEIKKEQLSGSPWILLRENEVSTLYKGEYHRAPVAIKVFKKLQAGSIAIVRQTFNKEIKTMKKFESPNILRIFGICIDETVTPPQFSIVMEYCELGTLRELLDREKDLTLGKRMVLVLGAARGLYRLHHSEAPELHGKIRSSNFLVTQGYQVKLAGFELRKTQTSMSLGTTREKTDRVKSTAYLSPQELEDVFYQYDVKSEIYSFGIVLWEIATGDIPFQGCNSEKIRKLVAVKRQQEPLGEDCPSELREIIDECRAHDPSVRPSVDEILKKLSTFSK

PTMs - Q8NB16 As Substrate

Site PTM Type Enzyme
K40 Ubiquitination
K50 Ubiquitination
S52 Phosphorylation
K57 Ubiquitination
T59 Phosphorylation
K66 Ubiquitination
K78 Ubiquitination
S92 Phosphorylation
S106 Phosphorylation
S125 Phosphorylation
S128 Phosphorylation
K157 Ubiquitination
S161 Phosphorylation
K173 Ubiquitination
K183 Ubiquitination
K198 Ubiquitination
K219 Ubiquitination
K230 Ubiquitination
T246 Phosphorylation
K249 Ubiquitination
T302 Phosphorylation
K331 Ubiquitination
S334 Phosphorylation
K354 Acetylation
K354 Ubiquitination
T357 Phosphorylation Q9Y572 (RIPK3)
S358 Phosphorylation Q9Y572 (RIPK3)
T364 Phosphorylation
K372 Ubiquitination
S373 Phosphorylation
T374 Phosphorylation
S393 Phosphorylation
S417 Phosphorylation
S467 Phosphorylation

Research Backgrounds

Function:

Pseudokinase that plays a key role in TNF-induced necroptosis, a programmed cell death process. Activated following phosphorylation by RIPK3, leading to homotrimerization, localization to the plasma membrane and execution of programmed necrosis characterized by calcium influx and plasma membrane damage. Does not have protein kinase activity. Binds to highly phosphorylated inositol phosphates such as inositolhexakisphosphate (InsP6) which is essential for its necroptotic function.

PTMs:

Phosphorylation by RIPK3 induces a conformational switch that is required for necroptosis. It also induces homotrimerization and localization to the plasma membrane.

Subcellular Location:

Cytoplasm. Cell membrane.
Note: Localizes to the cytoplasm and translocates to the plasma membrane on necroptosis induction.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Subunit Structure:

Homooligomer. Homotrimer; forms homotrimers on necroptosis induction. Interacts with RIPK3; the interaction is direct. Upon TNF-induced necrosis, forms in complex with PGAM5, RIPK1 and RIPK3. Within this complex, may play a role in the proper targeting of RIPK1/RIPK3 to its downstream effector PGAM5.

Family&Domains:

The protein kinase domain is catalytically inactive but contains an unusual pseudoactive site with an interaction between Lys-230 and Gln-356 residues. Upon phosphorylation by RIPK3, undergoes an active conformation (By similarity).

The coiled coil region 2 is responsible for homotrimerization.

Belongs to the protein kinase superfamily.

Research Fields

· Cellular Processes > Cell growth and death > Necroptosis.   (View pathway)

· Environmental Information Processing > Signal transduction > TNF signaling pathway.   (View pathway)

References

1). Cyclic helix B peptide alleviates proinflammatory cell death and improves functional recovery after traumatic spinal cord injury. Redox Biology, 2023 (PubMed: 37290302) [IF=11.4]
2). Polystyrene nanoplastics and cadmium co-exposure aggravated cardiomyocyte damage in mice by regulating PANoptosis pathway. Environmental pollution (Barking, Essex : 1987), 2024 (PubMed: 38462200) [IF=8.9]
3). An integrated network pharmacology approach reveals that Ampelopsis grossedentata improves alcoholic liver disease via TLR4/NF-κB/MLKL pathway. Phytomedicine : international journal of phytotherapy and phytopharmacology, 2024 (PubMed: 38981149) [IF=7.9]
4). Activation of ALDH2 attenuates high glucose induced rat cardiomyocyte fibrosis and necroptosis. Free radical biology & medicine, 2020 (PubMed: 31689484) [IF=7.4]

Application: WB    Species: rat    Sample: cardiomyocytes

Fig. 8 |Changes of RIP1, RIP3 and MLKL at mRNA and protein levels in the different groups (means ± SD, n=6) A, B, C: RIP1, RIP3 and MLKL mRNA levels in cardiomyocytes normalized by GAPDH levels. **P<0.01 compared with NG; #P<0.05, ##P<0.01 compared with HG; ^P<0.05, ^^P<0.01 compared with HG+Alda-1 D: Representative western blots of RIP1, RIP3 and MLKL proteins
5). Cyclic helix B peptide promotes random‐pattern skin flap survival via TFE3‐mediated enhancement of autophagy and reduction of ROS levels. British Journal of Pharmacology, 2022 (PubMed: 34622942) [IF=7.3]

Application: WB    Species: Mouse    Sample: skin tissues

FIGURE 2 CHBP inhibits necroptosis in random-pattern skin flaps. (a,b) Representative immunohistochemical staining for RIPK3 (brown) in mouse skin tissues from the control, FLAP and FLAP + CHBP groups and quantification of integrated absorbance of RIPK3 signal (n = 6 mice per group). The tissues were counterstained with haematoxylin (blue; scale bar = 100 μm). (c,d) Representative images of immunofluorescence staining of mouse skin samples (DAPI staining of the nuclei) and quantification graph for RIPK1 (green)-positive cells (n = 6 mice per group). Scale bar: 10 μm. Skin samples were harvested from mice in the control, FLAP and FLAP + CHBP groups. (e–g) CHBP treatment attenuated the activation of RIPK1, RIPK3 and MLKL induced by ischaemia–reperfusion injury. The expression of caspase 8 (CASP8) was significantly increased, which has an inhibitory effect on necroptosis. Densitometric quantification is shown (n = 6 mice per group). Data shown are means ± SEM. *P 
6). c-FLIP facilitates ZIKV infection by mediating caspase-8/3-dependent apoptosis. PLoS pathogens, 2024 (PubMed: 39038037) [IF=6.7]

Application: WB    Species: Human    Sample: HTR8 cells

S6 Fig Caspase-8 and caspase-3 involves in ZIKV replication. (A) HTR8 cells were infected with ZIKV at a MOI of 1 post sic-FLIP transfection. On D1 and D2 post-infection, MLKL, pMLKL and LC3B levels were measured by western blot. (B-C) Caspase-8 (B) and caspase-3 (C) were measured by qPCR post sicaspase-8 or sicaspase-3 transfection on day 1. (D-E) Western blot assays of caspase-8 (D) or caspase-3 (E) expression in HTR8 cells post sicaspase-8 or sicaspase-3 transfection for 1 day and 2 days. (F-I) HTR8 cells were infected with ZIKV at a MOI of 1 post sicaspase-8 (F-G) or sicaspase-3 (H-I) transfection for 24 hours. The viral load was measured on D1, D2 and D3 post-infection by qPCR (F, H) and plaque assay (H, I). The data represent either a single experiment chosen as representative from three independent experiments (B-C, F-I) or the collective results of three independent experiments (D-E). All the data are analyzed by unpaired Student’s t test. Data are presented as means ± SD. *P
7). 1, 3-Dichloro-2-propanol-Induced Renal Tubular Cell Necroptosis through the ROS/RIPK3/MLKL Pathway. Journal of Agricultural and Food Chemistry, 2022 (PubMed: 36000575) [IF=6.1]
8). Cetylpyridinium chloride triggers paraptosis to suppress pancreatic tumor growth via the ERN1-MAP3K5-p38 pathway. iScience, 2024 (PubMed: 39211547) [IF=5.8]
9). Quercetin-induced pyroptosis in colon cancer through NEK7-mediated NLRP3 inflammasome-GSDMD signaling pathway activation. American journal of cancer research, 2024 (PubMed: 38590424) [IF=5.3]

Application: WB    Species: Human    Sample: HCT116 and HT29 cells

Figure 4 Que-induced cell death independent of necroptosis pathway activation. A. Protein levels of MLKL and p-MLKL in HCT116 and HT29 cells treated with various doses of GSK’872 were measured using western blotting assay (n=3, where “n” represents the number of independent repeated experiments). Values are presented as mean ± SD. *P
10). Different cellular mechanisms from low- and high-dose zinc oxide nanoparticles-induced heart tube malformation during embryogenesis. Nanotoxicology, 2022 (PubMed: 36137004) [IF=5.0]
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