Phospho mlkl ser358

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-MLKL (Ser358) is a laboratory product that detects phosphorylation of the MLKL (Mixed Lineage Kinase Domain-Like Protein) protein at serine 358. MLKL is a key mediator of necroptosis, a form of programmed cell death.

Automatically generated - may contain errors

Lab products found in correlation

5 protocols using phospho mlkl ser358

Dissecting Apoptotic and Necroptotic Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following primary antibodies were used for Western blot: antibodies against survivin (#2803), XIAP (#14334), Caspase 8 (#9746S), Caspase 9 (#9508S), Caspase 3 (#9662S), cleaved-Caspase 3 (#9661S), PARP (#9542 L), ubiquitin (#3933S), RIP1 (#3493S), phospho-RIP1 (Ser166) (#44590), RIP3 (#13526S), phospho-RIP3 (Ser227) (#93654), phospho-MLKL (Ser358) (#91689S), TAB1 (#3226), TAK1 (#5206), phospho-TAK1 (Thr184/187) (#4508) and GAPDH (#5174) were from Cell Signaling Technology; anti-Caspase 7 antibody (#sc-28,295) was purchased from Santa Cruz and anti-MLKL antibody (#184718) was purchased from and Abcam. Z-VAD-fmk (Sigma-Aldrich, V116) and Nec-1 s (Biovision, 2535–1) were used to reveal apoptotic and necroptotic cell death, respectively. Cycloheximide (CHX, C7698-5G) was purchased from Sigma-Aldrich. MG132 (S2619) was obtained from Selleckchem.

Fang W., Che X., Li G., Wang A., Wang Y., Shi X., Hou K., Zhang X., Qu X, & Liu Y. (2020). Sur-X, a novel peptide, kills colorectal cancer cells by targeting survivin-XIAP complex. Journal of Experimental & Clinical Cancer Research : CR, 39, 82.

+ Open protocol

+ Expand

Protein Isolation and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total protein was isolated from cultured KCs and cellular supernatants using RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentration was measured by Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA) per manufacturer’s directions. 10-25ug protein was run on 4-12% precast polyacrylamide gels (Invitrogen) and transferred to 0.45µm polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk or BSA and incubated overnight at 4°C with primary antibodies (1:1000 dilution; XAF1 #13805; TRAIL #3219; IRF-1 #8478; Phospho-MLKL (Ser358) #91689; MLKL #14993; Cell Signaling Technology) followed by HRP-conjugated goat anti-rabbit IgG (1:10,000 dilution; sc-2301 Santa Cruz, Dallas, TX) or HRP-conjugated mouse anti-rabbit IgG (1:2000 dilution; sc-2357 Santa Cruz). Protein bands were detected by chemiluminescence using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and imaged by Omega Lum C (Gel Company, San Francisco, CA). Quantification was completed with ImageJ software relative to β-actin loading control (1:1000 dilution; #4967 Cell Signaling Technology).

Loftus S.N., Gharaee-Kermani M., Xu B., Moore T.M., Hannoudi A., Mallbris M.J., Klein B., Gudjonsson J.E, & Kahlenberg J.M. (2024). Interferon alpha promotes caspase-8 dependent ultraviolet light-mediated keratinocyte apoptosis via interferon regulatory factor 1. Frontiers in Immunology, 15, 1384606.

+ Open protocol

+ Expand

Analyzing Necroptosis Regulatory Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Nec-1 (HY-15760), Z-VAD(OMe)-FMK (HY-16658), Fer-1 (HY-100579), VX-765(HY-13205), 3-MA (HY-19312), Dox (HY-N0565A), AZA (HY-10586), and DEC (HY-A0004) were obtained from MedChem Express (Shanghai, China). SYTOX Green (S7020) was purchased from Life Technologies (CA, USA). R-2HG (SML2200) was from Sigma-Aldrich (Munich, German). Antibodies against RIPK1 (#3493), phospho-RIPK1 (Ser¹⁶⁶) (#65746), phospho-MLKL (Ser³⁵⁸) (#91689), MLKL (#14993), RIPK3 (#13526), phospho-RIPK3 (Ser²²⁷) (#93654), and H3K4me3 (#9751) were purchased from Cell Signaling Technology (MA, USA). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#60004-1-Ig), FLAG (#66008-4-Ig), and IDH2 (#66918-1-Ig) were obtained from Proteintech Group (Wuhan, China). The KDM2B antibody (#17-10264) was purchased from Sigma-Aldrich (Munich, German). Antibodies against IDH2R172K (ab264052) and H3 (ab1791) were obtained from Abcam (MA, USA).

Zhu S., Luo Y., Li K., Mei C., Wang Y., Jiang L., Wang W., Zhang Q., Yang W., Lang W., Zhou X., Wang L., Ren Y., Ma L., Ye L., Huang X., Chen J., Sun J, & Tong H. (2024). RIPK3 deficiency blocks R-2-hydroxyglutarate–induced necroptosis in IDH-mutated AML cells. Science Advances, 10(16), eadi1782.

+ Open protocol

+ Expand

Western Blot Analysis of Necroptosis Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

Briefly, ESCA cells under different treatments were rinsed with cold PBS two times and then lysed using RIPA buffer (Thermo Fisher Scientific, USA) containing protease inhibitors (Solarbio, Beijing, China) and phosphatase inhibitors (Solarbio, Beijing, China). Moreover, the contents of soluble proteins were determined by the BCA protein detection kit (Thermo Fisher Scientific, USA). Subsequently, Western blotting analysis was carried out as previously described [34 (link)] using antibodies against RIP (#3493), phospho-RIP (Ser166) (#65746), MLKL (#14993), phospho-MLKL (Ser358) (#91689), and GAPDH (#8884) (1:1,000; Cell Signaling Technology, Danvers, MA, USA).

Luo Z., Ding E., Yu L., Wang W., Guo Q., Li X., Wang Y., Li T., Zhang Y, & Zhang X. (2023). Identification of hub necroptosis-related lncRNAs for prognosis prediction of esophageal carcinoma. Aging (Albany NY), 15(11), 4794-4819.

+ Open protocol

+ Expand

Antibodies for Western Blotting and Flow Cytometry

Check if the same lab product or an alternative is used in the 5 most similar protocols

Antibodies for Western blotting were purchased from the following sources: Histone H2A (Cell Signaling Technology #3636); HMGB1 (Cell Signaling Technology #6893); β-Actin (Cell Signaling Technology #3700); Profilin-1 (Cell Signaling Technology #3237); Enolase-1 (Cell Signaling Technology #3810); Bip/GRP78 (Cell Signaling Technology #3177); Alpha-fodrin (EMD Millipore #MAB1622); MLKL (Cell Signaling Technology #14993); Phospho-MLKL Ser358 (Cell Signaling Technology #91689); GAPDH (ThermoFisher #AM4300). Antibodies for flow cytometry were purchased from the following sources: F4/80, PE-Cyanine5 (ThermoFisher #15-4801-82); CD11b APC-eFluor 780 (ThermoFisher #47-0112-82); CD11c Alexa Fluor 700 (ThermoFisher #56-0114-82); CD86 (B7-2) FITC (ThermoFisher #11-0862-82); CD40 Super Bright 436 (ThermoFisher #62-0401-82); MHC Class II I-Ab (ThermoFisher #17-5320-82); CD25 Alexa Fluor 488 (ThermoFisher #53-0251-82); CD69 Alexa Fluor 700 (BioLegend #104539).

Andersohn A., Garcia M.I., Fan Y., Thompson M.C., Akimzhanov A.M., Abdullahi A., Jeschke M.G, & Boehning D. (2019). Aggregated and Hyperstable Damage-Associated Molecular Patterns Are Released During ER Stress to Modulate Immune Function. Frontiers in Cell and Developmental Biology, 7, 198.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!