γ 32p labeled atp

Media and salts were purchased from Thermo Fisher, USA. Glycine, imidazole, and ATP from Merck Sigma-Aldrich, USA. Protein marker from Thermo Scientific, USA. Antibiotics, isopropyl β-D-1-thiogalactopyranoside (IPTG) and dithiothreitol (DTT) from GoldBio Inc, USA. Protease inhibitor cocktail from Amresco, USA. Ni²⁺-NTA resin from Qiagen, GmBH. γ³²P-labeled ATP (>3000 Ci/mmol) from Perkin Elmer, USA.

rhIKKβ was purchased from Merck Millipore, Burlington, MA, USA; and rhTAK1-TAB1 from Promega, Madison, WI, USA. These catalytically active enzymes were reacted with major basic protein (MBP) as the exogenous substrate in the presence of 5 μCi [γ-³²P]-labeled ATP (Perkin Elmer) at 30 °C for 30 min. The reaction mixtures were spotted onto a P81 phosphocellulose filter, washed with 0.75% H₃PO₄, and washed again with 100% acetone. Kinase activity was measured as count per minute (cpm) of radioactivity.

All peptides were labeled at 10 µM or 50 µM final concentrations as follows. Peptides were diluted in 10× NEBuffer for Protein Kinases (New England Biolabs) followed by the addition of 40 µM γ³²P-labeled ATP (Perkin Elmer) and protein kinase A (2,500 units) and incubation at 32 °C for 2 h. Proteins and peptides labeled at 50 µM or above were first incubated with radio-labeled ATP for 1 h followed by the addition of unlabeled ATP (400 µM) for an additional 1 h. Donor ubiquitins were similarly labeled at a concentration of 200 µM, whereas BRD4 was labeled at 50 µM.

The San1 peptide was radiolabeled (50 µM) in the presence of γ-³²P labeled ATP (Perkin Elmer; Waltham, MA, USA) and cAMP-dependent Protein Kinase (New England Biolabs; Ipswich, MA, USA) for 1 h at 30 °C in a reaction buffer that had been supplemented with tween-20 (0.1%). All reactions were performed in a buffer containing 30 mM Tris, pH 7.5, 5 mm MgCl₂, 2 mM ATP, 2 mM DTT, and 0.1% Tween-20. Human E1 (1 µM), WT ubiquitin (60 µM), Ubc1 (10 µM), and either full-length San1 or San1_1–303 (0.5 µM) were sequentially added to Eppendorf tubes and incubated for 2 min at room temperature. Next, 3 µM radiolabeled San1 peptide, 3 µM radiolabeled San1 peptide mixed with 3 µM unlabeled San1 peptide, or 3 µM radiolabeled San1 peptide mixed with 6 µM unlabeled San1 peptide were then added to initiate the respective ubiquitylation reactions. Reactions were quenched at various time-points in 2X SDS-PAGE buffer and substrate and ubiquitylated products were separated by SDS-PAGE on 4–20% gels (Lonza; Basel, Switzerland). Gels were dried and exposed to phosphor screens for autoradiography. The quantification of substrates and products was performed as described in the limited proteolysis section. The fraction of ubiquitylated San1 peptide was calculated by dividing the amount of peptide that had been modified by one or more ubiquitins by the total signal in the lane.