Ubiquitin

Extract was treated with buffer, with 50 μM ubiquitin (Boston Biochem, U100-H) for a combination of 50 μM ubiquitin and 10 μM UbVS. Samples were collected after 30 minutes. Dried peptides (2 mg of proteins) were resuspended in IAP buffer [50 mM MOPS (pH 7.2), 10 mM sodium phosphate and 50 mM NaCl] and centrifuged at 15000 RPM (5 min). After that the supernatants were added to the diGly resin (Cell signaling, 5562) and incubated for 2 hr at 4°C. After that, beads were washed three times with ice-cold IAP buffer and twice with PBS. The diGly peptides were eluted twice with 0.15% TFA, desalted using homemade StageTips and dried via vacuum centrifugation. Peptides were immunoprecipitated twice. Samples were processed for Mass Spectrometry analysis by SL-TMT method (described below).

For the in vivo ubiquitination assay, 293T cells were transfected with the indicated plasmids for 36 h and lysed in ice-cold lysis buffer TNTE 0.5% (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 0.5% Triton X-100, containing 10 mM NaF, 2 mM Na3VO4, 10 mg/ml leupeptin and 1 mM PMSF). The cell lysates were then subjected to anti-Flag IP, eluted by boiling 10 min in 1% SDS, diluted 10 times in lysis buffer TNTE 0.5% and then re-immunoprecipitated with anti-Flag antibody (2 × IP). The ubiquitin-conjugated proteins were detected by immunoblotting with appropriate antibodies. For in vitro ubiquitination reactions, bacterially expressed GST-Rac1-Flag and GST-TRAF6 proteins were purified using glutathione sepharose beads in TNTE 0.5% buffer. ubiquitination reactions were carried out with 0.3 mg Rac1, 0.35 mg rabbit E1 ubiquitin activating enzyme (Boston Biochem, Cambridge, MA, USA), 0.4 mg UBE2N/UEV1A recombinant E2 complex (Boston Biochem), and 10 mg of ubiquitin (Boston Biochem) in 20 μl of ubiquitin conjugation reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 0.05 mM DTT, supplemented with 5 mM ATP). Rac1 was loaded with GTPγS or GDP or left in its Mg²⁺-free GDP/GTP-empty form, as described previously ^{50 (link)}.

The ubiquitylation of CBP was performed in a reaction mixture containing synthesized WT or mutant CBP protein in 50 mM tris (pH 7.6), 5 mM MgCl₂, 0.6 mM dithiothreitol (DTT), 2 mM adenosine triphosphate, E1 enzyme (1.5 ng/μl, Boston Biochem), Ubc5 (10 ng/μl), Ubc7 (10 ng/μl), ubiquitin (1 μg/μl, Boston Biochem), 1 μM ubiquitin aldehyde, and His-purified recombinant Cullin 1, Skp1, Rbx1, and synthesized FBXL19. The reaction mixture was then subjected to Western blotting analysis with anti-ubiquitin antibody.