Protein extracts were centrifuged and the pellets were collected and subjected to SDS-PAGE to purify the proteins. The bands obtained were resolved on a 12% Tris-buffered saline (TT), SDS-PAGE gels (Bio-Rad, Hercules, CA) to separate the bands. Proteins were transferred onto polyvinylidene difluoride membranes with 2% Triton X-100, 100 MML/L Trisha (pH 7.4), 100 MML/L NaCl (pH 7.4) and 100% phosphatase for 1 h at RT. Primary Western Blots were incubated overnight at 4 °C with a horseradish peroxidase-conjugated rabbit anti-human IgG1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), an Alexa-Fluor 488 goat anti-mouse IgG1 antibody (Santa Cruz Biotechnology, Inc.), or a rabbit anti-rabbit IgG1 antibody (Cell Signaling Technology, Burlington, CA, CV1125-01), incubated in blocking buffer (pH 7.4) at room temperature for 1 h. The blot was then incubated with a secondary antibody conjugated with horseradish peroxidase-conjugated goat anti-rabbit (Abram, Cambridge, MA), followed by scanning with enhanced chemiluminescence detection (ECL, GE Healthcare, Rockford, IL, USA) for 45 min at 50 V (GE Healthcare). The immunoblots were analyzed with chemiluminescence detection kit (GE Healthcare). Antibodies detected by the ECL kit were visualized with enhanced chemiluminescence detection (GE Healthcare) at 490 nm. Immunohistochemically-identified bands are included and are included in the Supplementary Table S1.
Acknowledgments The authors thank T. Schmitt for assistance with the structure of the protein. We thank Dr. H. Hole for help with the structure of A. catalysis. This work was supported by the University of Tübingen and the German Research Foundation, and by Disc and the German Research Council (DKK).
Author Contributions Conceived and designed the experiments: NBS CAB. Performed the experiments: NBS. Analyzed the data: NBS. Contributed reagents/materials/analysis tools: CAB.
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N my interactor protein ab103504 1 Product Data sheet 1 Overview Product name N my interactor protein description Recombinant full length Human N my interactor (amino acids 1-307) with an N terminal
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