Co-Immunoprecipitation of Signaling Proteins

Samples were harvested in CHAPS buffer and subjected to co-immunoprecipitation (co-IP) using Dynabeads magnetic protein A agarose beads (Life Technologies) according to the manufacturer’s protocol. Immunoprecipitation antibodies were crosslinked to the beads using the Pierce BS3 crosslinker (ThermoFisher, cat. 21580). 1 mg of protein lysate was used as input material for co-IP reactions. Antibodies were acquired from Cell Signaling Technology unless noted otherwise. Antibodies were used for MEK (#8727), ERK (#9102), pMEK (#9154), pERK (#4376), BCL-2 (#4223), BCL-XL (#2764), MCL-1 (#4572), BIM (#2933), α-tubulin (#3873) and β-actin (Abcam, Ab6276).

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Eleveld T.F., Vernooij L., Schild L., Koopmans B., Alles L.K., Ebus M.E., Dandis R., van Tinteren H., Caron H.N., Koster J., van Noesel M.M., Tytgat G.A., Eising S., Versteeg R., Dolman M.E, & Molenaar J.J. (2023). MEK inhibition causes BIM stabilization and increased sensitivity to BCL-2 family member inhibitors in RAS-MAPK-mutated neuroblastoma. Frontiers in Oncology, 13, 1130034.

Publication 2023

Actin Agarose Antibodies Bcl 2 Buffer Chaps Co immunoprecipitation Immunoprecipitation Protein Protein a α tubulin

Corresponding Organization :

Other organizations : Princess Máxima Center, Cancer Genomics Centre, Roche (Switzerland), Utrecht University

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Variable analysis

independent variables

Immunoprecipitation antibodies

dependent variables

Protein levels of MEK
Protein levels of ERK
Phosphorylation levels of MEK
Phosphorylation levels of ERK
Protein levels of BCL-2
Protein levels of BCL-XL
Protein levels of MCL-1
Protein levels of BIM

control variables

Protein concentration of lysate used for co-IP (1 mg)
Dynabeads magnetic protein A agarose beads used for co-IP
Pierce BS3 crosslinker used to crosslink antibodies to beads
α-tubulin and β-actin as loading controls

positive controls

None specified

negative controls

None specified

Annotations

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