293 T cells were independently transfected with plasmids encoding MYC-ULK1 or FLAG-TRIM32. Forty-eight hours later, cells were lysed in RIPA buffer for ULK1 purification and tris buffer for TRIM32 immunoprecipitation, both containing protease inhibitors, as previously indicated. Lysates were cleared by centrifugation and subjected to immunoprecipitation for 2 hours using agarose-coupled antibodies against MYC or FLAG tags. TRIM32 protein was eluted for 1 hour by means of FLAG peptide (Sigma-Aldrich, F3290) at 400 ng/μl in the 1X Ubiquitin Conjugation Reaction Buffer (Boston Biochem, B-70). The ubiquitination assays were performed in 100 μl of reaction volume, combining 20 μl of immunopurified MYC-ULK1 bound to agarose-beaded Myc antibody, 20 μl of eluted TRIM32, and the following recombinant components: 100 nM E1 Ub–activating enzyme (Ube1; Boston Biochem, E-305), 1 μM E2 Ub–conjugating enzyme (Ube2N; Boston Biochem, E2-664), and 50 μM HA-ubiquitin (Boston Biochem, U-110) resuspended in Ubiquitin Reconstitution Buffer (Boston Biochem, B-90). In some in vitro ubiquitination experiments, recombinant ULK1 protein (SignalChem, U01-11G) was used instead of the immunopurified one. The reaction was performed in 1X Ubiquitin Conjugation Reaction Buffer, supplemented with Mg2+–adenosine triphosphate (Boston Biochem, B-20) at 2 mM and incubated at 30°C for 2 hours in a rocking platform. When indicated, a denaturation step was added as previously described, followed by a reimmunoprecipitation of ULK1 using the rabbit anti-ULK1 H240 antibody (Santa Cruz Biotechnology, sc-33183). The incorporation of ubiquitin was analyzed by immunoblotting using rabbit anti-HA antibody (Sigma-Aldrich, H6908) to detect HA-ubiquitin.

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