Immunoprecipitation of RIPK1 Phosphorylation

To monitor phosphorylation and ubiquitination of RIPK1, we used TNF-Flag treatment.28 (link) Liver tissue was homogenised in buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-Potassium hydroxide pH7.5, 0.2% NP-40, 120 mM NaCl, 0.27M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 2 mM Na3VO4, cOmpleteTM Protease Inhibitor Cocktail, 2 mM phenylmethylsulfonyl fluoride and 10 mM N-Ethylmaleimide. Samples were either incubated with anti-FLAG M2 magnetics beads (Sigma) for 4 hours or RIPK1 antibody (Cell Signaling) overnight followed by 2 hours incubation with magnetics beads (Bio-rad) at 4°C. The beads were washed and proteins were eluted in 70°C with sample buffer and the eluted proteins were fractionated by SDS-PAGE gels. Proteins were detected by immunoblotting as described above.

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Zhuang Y., Xu H.C., Shinde P.V., Warfsmann J., Vasilevska J., Sundaram B., Behnke K., Huang J., Hoell J.I., Borkhardt A., Pfeffer K., Taha M.S., Herebian D., Mayatepek E., Brenner D., Ahmadian M.R., Keitel V., Wieczorek D., Häussinger D., Pandyra A.A., Lang K.S, & Lang P.A. (2019). Fragile X mental retardation protein protects against tumour necrosis factor-mediated cell death and liver injury. Gut, 69(1), 133-145.

Publication 2019

anti-FLAG M2 magnetics beads RIPK1 antibody

Acid Antibody Buffer Edta Egta Ethylmaleimide Gels Liver Nacl Np 40 Phenylmethylsulfonyl fluoride Phosphorylation Potassium hydroxide Protease inhibitor Proteins Ripk1 Sds page Sodium pyrophosphate Sucrose Tissue Ubiquitination β glycerophosphate

Corresponding Organization : Heinrich Heine University Düsseldorf

Other organizations : Düsseldorf University Hospital, National Research Centre, University of Southern Denmark, Odense University Hospital, Luxembourg Institute of Health, University of Luxembourg, University of Duisburg-Essen

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Variable analysis

independent variables

TNF-Flag treatment

dependent variables

Phosphorylation of RIPK1
Ubiquitination of RIPK1

control variables

Buffer composition (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-Potassium hydroxide pH7.5, 0.2% NP-40, 120 mM NaCl, 0.27M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 2 mM Na3VO4, cOmpleteTM Protease Inhibitor Cocktail, 2 mM phenylmethylsulfonyl fluoride and 10 mM N-Ethylmaleimide)
Temperature (4°C)
Incubation time (4 hours for anti-FLAG M2 magnetic beads, overnight for RIPK1 antibody followed by 2 hours for magnetic beads)

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