Immunoprecipitation of c-Myc Protein

The procedure was performed as described previously (Shimmoto et al, 2009 (link)). For immunoprecipitation, ∼1.0 × 10⁸ cells from 50 ml culture were harvested and washed once with PBS. The cells were then re-suspended in 0.5 ml of IP buffer (20 mM HEPES-KOH [pH 7.6] [Nacalai tesque], 50 mM potassium acetate [Sigma-Aldrich], 5 mM magnesium acetate [FUJIFILM Wako], 0.1 M sorbitol [FUJIFILM Wako], 0.1% TritonX-100 [Sigma-Aldrich], 2 mM DTT [FUJIFILM Wako], 20 mM Na₃VO₄ [Sigma-Aldrich], 50 mM β-glycerophosphate [Sigma-Aldrich], and Protease Inhibitor Cocktail [Sigma-Aldrich]) and were disrupted with glass beads using a multi-beads shocker (Yasui Kikai). The lysates were cleared by centrifugation (20,000g for 10 min at 4°C). The supernatants of lysates were mixed with anti-c-Myc antibody (Nacalai tesque) attached to Protein G Dynabeads (10004D; Thermo Fisher Scientific). After incubating for 1 h, the beads were washed with IP buffer and proteins were extracted by boiling with 1× sample buffer (2% SDS [Nacalai Tesque], 4 M Urea [Nacalai Tesque], 60 mM Tris–HCl [pH 6.8] [Nacalai Tesque], 10% Glycerol [Nacalai Tesque], and 70 mM 2-mercaptethanol [Sigma-Aldrich]).