Nano pure system

All reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless stated otherwise. Formic acid (FA) and glacial acetic acid (HOAc) were purchased from Fisher Scientific (Pittsburgh, PA). Methanol was purchased from Honeywell Burdick & Jackson (Wicklow, Ireland). Water was deionized by a NanoPure system from Thermo Scientific (Marietta, OH). Fused silica capillary tubing was purchased from Polymicro Technologies (Phoenix, AZ). The RPLC column (Jupiter 5 μm C5 300 Å, LC Column 250 × 4.6 mm) was purchased from Phenomenex (Torrance, CA).

Bovine pancreas TPCK-treated trypsin, urea, ammonium bicarbonate (NH₄HCO₃), dithiothreitol (DTT), iodoacetamide (IAA), cOmplete™ Protease Inhibitor Cocktail and PhosSTOP™ tablets (provided in EASYpacks), Microcon-30kDa Centrifugal Filter Unit with Ultracel-30 membrane (molecular weight cut-off, MWCO: 30 kDa, RC30 kDa) were purchased from Sigma-Aldrich (St. Louis, MO). Ultracel regenerated cellulose (RC) membranes with 100 kDa MWCO (RC100 kDa), Biomax polyethersulfone (PES) membranes with 50 kDa MWCO (PES50 kDa), 100 kDa MWCO (PES100 kDa), 300 kDa MWCO (PES300 kDa), 500 kDa MWCO(PES500 kDa), and Durapore hydrophilic polyvinylidene fluoride (PVDF) membrane with 0.1 μm pore size (HILICPVDF 0.1 μm) were purchased from MilliporeSigma (Burlington, MA). Formic acid (FA) and acetonitrile (ACN) were purchased from Fisher Scientific (Pittsburgh, PA). Methanol was purchased from Honeywell Burdick & Jackson (Wicklow, Ireland). MS compatible human protein extract (Intact K562 human cell lysate) was purchased from VWR (Radnor, PA). Mammalian Cell-PE LBTM buffer for cell lysis was purchased from G-Biosciences (St. Louis, MO). Water was deionized by a Nano Pure system from Thermo Scientific (Marietta, OH).

Xenopus laevis animals were purchased from Nasco (Fort Atkinson, WI USA). Animals were housed in a pathogen-free environment on a 12 h light – 12 h dark schedule. NP40 was purchased from G-Biosciences (St. Louis, MO USA). 1,1,2 trichloro 1,1,2 trifluroethane (Freon) was purchased from AquaSolutions (Deer Park, TX USA). NuPage 4–12% Bis-Tris Gel, MOPS SDS Running Buffer, SimplyBlue SafeStain^™, and LDS Samper Buffer were purchased from Novex (Carlsbad, CA USA). Complete, mini protease inhibitor cocktail, provided in EASYpacks, was purchased from Roche Diagnostics (Indianapolis, IN USA). Human Chorionic Gonadotropin (HCG), bovine pancreas TPCK-treated trypsin, urea, NH₄HCO₃, dithiothreitol (DTT), iodaoacetamide (IAA), cysteine, SDS, and acetone were purchased from Sigma-Aldrich (St. Louis, MO USA). Water was deionized by a Nano Pure system from Thermo scientific (Marietta, OH). Acetonitrile (ACN), formic acid (FA), Pierce C-18 Spin Columns, and Pierce BCA Protein Assays were purchased from Thermo Scientific (Marietta, OH USA). Sep-Pak Vac (1 cc, 100 mg) C-18 Cartridges were purchased from Waters (Ireland). Centrifugal Filter Units with a 30,000 NMWL cut-off were purchased from Millipore (Carrigtwohill, CO USA). Marc’s Modified Ringers (MMR) and MBS were made in-house (See Electronic Supplemental Material (ESM) page S-10).