Figure 1: Structure of the MLKL target protein.

MLKL Introduction

Protein Function

• MLKL plays a crucial role in TNF-induced programmed cell necrosis.
• MLKL belongs to the protein kinase superfamily, containing a pseudo-kinase domain that lacks catalytic activity.
• MLKL is activated upon phosphorylation by RIPK3, localizing to the plasma membrane to execute necroptosis characterized by calcium influx and plasma membrane damage.
• In addition to TNF-induced necroptosis, nuclear necroptosis can also occur. Upon activation by ZBP1, MLKL is phosphorylated by RIPK3 in the nucleus, leading to nuclear membrane disruption and leakage of cellular DNA into the cytoplasm.

Protein Characteristics

• MLKL is activated upon phosphorylation by RIPK3, localizing to the plasma membrane to execute necroptosis characterized by calcium influx and plasma membrane damage.
• In addition to TNF-induced necroptosis, nuclear necroptosis can also occur. Upon activation by ZBP1, MLKL is phosphorylated by RIPK3 in the nucleus, leading to nuclear membrane disruption and leakage of cellular DNA into the cytoplasm.
• The interaction between MLKL and RIPK3 is species-specific: human MLKL only interacts with human RIPK3, not mouse RIPK3.
• Phosphorylated MLKL (p-MLKL) is a trigger for necroptosis. It is absent in normal tissues and can only be detected in infected, damaged, or aging tissues.

Protein Expression

• MLKL forms a homotrimer upon activation.
• Phosphorylated MLKL (p-MLKL) triggers necroptosis and is not detected in normal tissues but can be observed in infected, damaged, or aging tissues.
• Detection of p-MLKL typically requires induction of necroptosis stimuli (commonly TSZ-TNFα + Smac mimetic + z-VAD).

Protein Localization

• MLKL is primarily located in the cytoplasm and translocates to the plasma membrane during necroptosis.
• It localizes to the nucleus in response to certain viral infections.

Figure 2: MLKL ICC Experimental Results Image, Recombinant Alexa Fluor® 488 Anti-MLKL antibody [EPR17514] (ab207901). Green: MLKL; Red: Tubulin

Isoforms & Post-Translational Modifications

• Human (Q8NB16): Isoforms 1-2; 30-54 kDa (predicted)
• Mouse (Q9D2Y4): Isoforms 1-2; 53-54 kDa (predicted)
• Phosphorylation modifications.
• Can be inhibited by necrosulfonamide (NSA), an inhibitor of necroptosis.
• Activation of MLKL not only requires phosphorylation by RIPK3 but also binding to highly phosphorylated phosphoinositides.

WB Experiment Tips

Precautions

• To detect p-MLKL, samples need to be induced with necroptosis stimuli such as TSZ (TNFα + Smac mimetic + z-VAD) treatment.
• We recommend checking upstream and downstream markers in the MLKL signaling pathway to confirm successful induction.
• When detecting phosphorylation modifications, confirm the total MLKL protein content in the cells first.
• Use freshly prepared samples to prevent the weakening of phosphorylation modifications due to frozen storage and add appropriate phosphatase inhibitors.
• Depending on the protein modification, we recommend using suitable positive controls.
• If protein signals are weak when using conventional lysis methods, consider preparing cell lysates using the 1% SDS heated lysis method.

Positive Control

• MLKL: Huvec and HeLa cell lysates.
• p-MLKL: HT-29 cell lysate treated with TNFα + Smac mimetic + z-VAD for 8 hours.

Negative Control

• Human MLKL knockout HeLa cell line (ab255408) / cell lysate (ab263788).

Example Results

Figure 3: WB-Recombinant Anti-MLKL Antibody [EPR17514] (ab184718)

Lane 1: HUVEC cell lysate
Lane 2: HT-29 cell lysate
Lane 3: Wild-type HeLa cell lysate
Lane 4: MLKL knockout HeLa cell lysate

Results Description: MLKL (green), GAPDH (red)

Predicted Band Size: 54 kDa

Figure 4: WB-Recombinant Anti-MLKL (phospho S345) Antibody [EPR9515(2)] (ab196436)

Lane 1: Untreated L-929 (mouse fibroblast-like cells from connective tissue) whole cell lysate.
Lane 2: L-929 cells treated with 20 ng/ml TNFα (ab9642), 100 nM Smac mimetic, and 20 µM z-VAD (ab120382) for 8 hours, then harvested for whole cell lysate.

Detected Band Size: 54 kDa

 
Key control point

In addition to the general issues that need to be paid attention to during the experiment, special attention should be paid to the following key control points:

Sample preparation:

1. Add complex protease inhibitors to avoid target protein degradation.
2. Keep samples on ice throughout sample preparation.
3. Determine the total protein concentration of the sample by Bradford analysis, Lowry analysis or BCA analysis.

Transfer film:

1. It is recommended to use Ponceau staining after the transfer is completed to determine whether the transfer was successful.

Antibody incubation:

1. Please select the appropriate antibody working concentration according to the product instructions.
2. It is recommended to use freshly prepared antibodies and not reuse them.
3. It is recommended that primary and secondary antibodies be diluted in blocking solution.
4. It is recommended to add a control with only the secondary antibody and no primary antibody.

References

1. Liming Sun, Huayi Wang, Zhigao Wang, Sudan He, She Chen, Daohong Liao, Lai Wang, Jiacong Yan, Weilong Liu, Xiaoguang Lei, Xiaodong Wang. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell. 2012 Jan 20;148(1-2):213-27. doi: 10.1016/j.cell.2011.11.031.
2. Zhigao Wang, Hui Jiang, She Chen, Fenghe Du, Xiaodong Wang. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell. 2012 Jan 20;148(1-2):228-43. doi: 10.1016/j.cell.2011.11.030.
3. Zhenyu Cai, Siriporn Jitkaew, Jie Zhao, Hsueh-Cheng Chiang, Swati Choksi, Jie Liu, Yvona Ward, Ling-Gang Wu, Zheng-Gang Liu. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol. 2014 Jan;16(1):55-65. doi: 10.1038/ncb2883. Epub 2013 Dec 8.
4. Lorenzo Galluzzi, Oliver Kepp, Francis Ka-Ming Chan, Guido Kroemer. Necroptosis: Mechanisms and Relevance to Disease. Annu Rev Pathol. 2017 Jan 24;12:103-130. doi: 10.1146/annurev-pathol-052016-100247. Epub 2016 Dec 5.
5. Junying Yuan, Palak Amin, Dimitry Ofengeim. Necroptosis and RIPK1-mediated neuroinflammation in CNS diseases. Nat Rev Neurosci. 2019 Jan;20(1):19-33. doi: 10.1038/s41583-018-0093-1.