Denaturing immunoprecipitation for in vivo ubiquitination assay

To study protein ubiquitination, denaturing immunoprecipitations were performed as described previously (Jang et al., 2015 ). Briefly, cells were lysed in denaturing buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% SDS, 1 mM Na₃VO₄, 10 mM NaF, 5 mM NEM, 10 μM MG132) and boiled for 10 min. Lysates were diluted 1:10 with the same buffer without SDS and incubated with the appropriate antibody overnight with rotation at 4°C. Protein G-­agarose was added, and the agarose beads were washed four times in lysis buffer (without SDS). Proteins were eluted at 95°C in SDS loading buffer, separated by SDS–PAGE, and transferred to nitrocellulose membrane.