G a beads

GFP-Cdt 2–345 (Fig. 1 A) was immunoprecipitated from lysates of transiently transfected U2OS cells by anti-GFP antibodies (Roche) bound to G/A beads (Santa Cruz Biotechnology, Inc.). After three washes and elution from the beads, GFP-Cdt 2–345 and coprecipitated proteins were subsequently digested with trypsin and Lys-C. Samples were desalted on C-18 stage tips (Nest Group) and analyzed by nanoflow HPLC tandem mass spectrometry (2D-NanoLC; Eksigent; Orbitrap Velos mass spectrometry; Thermo Fisher Scientific; Vasilj et al., 2012 (link)). Identification of proteins was performed with Mascot (V2.2; Matrix Science), and isoform specificity of peptides was obtained from the Proteomicsdb database (Wilhelm et al., 2014 (link)).

For Western blot analysis, cells were lysed in Triton X-100–based (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% Triton X-100, and Roche protease inhibitor cocktail) or hypotonic (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, and Roche protease inhibitor cocktail) lysis buffer as indicated in figure legends. For IP, Triton X-100–containing lysates were supplemented with indicated antibodies and G/A beads (Santa Cruz Biotechnology, Inc.) and rotated at 4°C. Beads were washed with low-salt NET buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, and 1% Triton X-100), and proteins were eluted from the beads. Proteins were separated according to sizes by SDS-PAGE and blotted on a nitrocellulose membrane (VWR), which was probed with indicated antibodies. Antibodies were purchased from the following distributors: Abcam, rb α Pgam5 (ab126534); BD, m α β-catenin (610153); Cell Signaling Technology, rb α axin1 (C76H11), m α Myc (9B11), rb α phospho–β-catenin (Ser33/37); Roche, m α GFP (11814460001), Santa Cruz Biotechnology, Inc., rb α β-catenin (H-102); AbD Serotec, r α α-tubulin (MCA77G); and Sigma-Aldrich, rb α Flag (F7425), m α GST (G1160), rb and m α TOM20 (HPA011562/MABT166), and m α β-actin (A5441). Intensities of Western blot bands were quantified with AIDA 2D densitometry.