FLAG-tagged Xenopus bap1 mRNA was synthesized from Not I–linearized pCS2+ plasmids using mMESSAGE mMACHINE SP6 transcription kit (Thermo Fisher Scientific, Waltham, MA). Embryos were injected at the one-cell stage with 0.5 ng of the synthetic mRNA and maintained at 22°C until they reached stage 12. Then, ~250 injected embryos were collected and lysed in 2.5 ml of mammalian cell PE LB buffer (G-Biosciences, St. Louis, MO) containing a complete protease inhibitor cocktail (Roche, Indianapolis, IN) by passing embryos through a 21-gauge needle ~20 times, placed on ice for 30 min, and then centrifuged at 12,000g for 10 min to obtain clear lysates. An equal number of uninjected sibling embryos were processed in an identical fashion, and then 1× FLAG peptide (Sigma-Adrich, St. Louis, MO) was added to the clear lysate to the final concentration of 10 ng/ml to be used as a control. Both FLAG-Bap1 and FLAG-containing control lysates were precleared by coincubation with magnetic protein G beads (Thermo Fisher Scientific, Waltham, MA) and mouse immunoglobulin G (2 μg/ml) at 4°C for 2 hours. Final coimmunoprecipitation was performed overnight at 4°C using FLAG-M2 mouse antibody, followed by a 10-min wash with coimmunoprecipitation buffer. The magnetic beads containing coimmunoprecipitated complexes were sent for further processing and analysis to the Proteomics and Metabolomics Shared Resource at The Wistar Institute (Philadelphia, PA). Mass spectrometry findings were filtered in the following manner. Any protein with a label-free quantification (LFQ) intensity greater in the control than the FLAG-Bap1 sample was discarded. In addition, all proteins were removed where the control LFQ intensity was >1% of the corresponding FLAG-Bap1 LFQ intensity. Furthermore, proteins were required to have at least an LFQ intensity of 5,000,000 and two unique peptide reads. Last, results were filtered for known mass spectrometry contaminants of FLAG-tagged experiments using the CRAPome (www.nature.com/articles/nmeth.2557).

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