Ube2h5b

For cCBL phosphorylation, 50 ng of Y505F-Lck [18 (link)] were mixed with the cCBL IP (1 mg cell extract) or r-(recombinant) cCBL (200 ng) and incubated in Lck kinase buffer (25 mM Tris-HCl pH 7.4, 5 mM MnCl₂, 50 μM ATP) for 15 min at 25 °C. cCBL reactions were performed in ubiquitination buffer (40 mM Tris-HCl pH 7.4, 2 mM DTT, 5 mM MgCl₂, 5mM ATP). The reaction volumes (40 μL) contained 200 ng purified recombinant (r)-cCBL (Merck-Millipore, Burlington, MA, USA), 5 μg ubiquitin, 250 ng UBA1, and 250 ng of UBE2H5B or UBE2N/Ubc13 (all from Boston Biochem, Cambridge, MA, USA). Parallel reactions with cCBL and WWP1 (20 μL) were performed in ubiquitination buffer using r-cCBL (200 ng) or r-WWP1 (Ubiquigent, Dundee, Scotland, UK) (200 ng), UBE2H5B (300 ng), UBA1 (50 ng), and energy regeneration solution (Boston Biochem). To test for PTEN ubiquitination, GST-PTEN (100–400 ng, indicated) was purified by pull-down, suspended in ubiquitination buffer, and added to the reaction. Reactions (2 h, 30 °C) were terminated with 1× Laemli buffer.