Anti myc 9e10 sc 40

An in vivo ubiquitination assay was performed as described previously.23 (link) HEK-293T cells were transfected with the vectors described above, and 2 days after transfection, cells were lysed in boiling buffer (1% SDS in PBS). After heating the lysate for 5 min at 100°C and sonicating to shear the DNA, immunoprecipitations (IP) were performed in 1% Triton X-100, 0.5% SDS, 0.25% sodium deoxycholate, 0.5% BSA, 1 mM EDTA in PBS containing a protease inhibitor cocktail (Sigma, St. Louis, MO, USA) using anti-HA (HA.11 Monoclonal Antibody #MMS-101R; Covance, Princeton, NJ, USA) or anti-BAP1 antibody (sc-28383, Santa Cruz, Dallas, TX, USA) and Protein G Sepharose beads (GE Healthcare, Little Chalfont, UK). Reaction mixtures were incubated at 4°C for 16 h and protein beads were washed twice with the same buffer and twice with 10-fold diluted PBS. Samples were subjected to SDS-PAGE and immunoblotted using anti-Myc (9E10 sc-40, Santa Cruz), anti-FLAG (monoclonal anti-FLAG M2 antibody, Sigma), anti-HA (anti-HA high affinity#1867423, Roche Applied Science) and anti-BAP1 antibodies. Experiments were independently repeated in triplicate and ubiqitination level was quantified by Image Quant TL (GE Healthcare).