Nebuffer for protein kinase

rFMRP was diluted to 11.7 µm in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 mm ATP with or without active CK2 (New England Biolabs, #P6010). Reactions (25-µl) were performed according to the manufacturer’s protocol (New England Biolabs). Human recombinant FMRP S500 was generated as previously described (Evans and Mihailescu, 2010 (link)).

The time-course of phosphorylation of 30 μM abltide by 0.2 U/mL human active Abl kinase (1067 U/mg, Merck Millipore) and [T315I]Abl (Abl(T315I) protein, Merck Millipore) was analyzed using Liquid Chromatography-Mass Spectrometry (LC/MS, Shimadzu LCMS-2020). Abltide and Abl kinase were prepared and mixed in kinase buffer (50 mM Tris-HCl, 10 mM MgCl₂, 0.1 mM EDTA, 2 mM dithiothreitol and 0.01% Brij 35 (v/v), pH 7.5; NEBuffer for protein kinases, New England Biolabs) at a desired concentration and the reaction was initiated by the addition of 500 μM ATP (adenosine 5′-triphosphate disodium salt hydrate, Sigma-Aldrich). The mixture was incubated at 37 °C with gentle shaking and the reaction was terminated with 10% 20 mg/mL dihydroxybenzoic acid at nine time points (0, 5, 10, 20, 30, 45, 60, 90 and 120 min). Samples were injected onto a C₁₈ analytical column (Jupiter 300 5 μm 300 Å, 150 × 2.0 mm, Phenomex) and the mass of each component was evaluated using MS. The percentage of phosphorylation of abltide was determined by comparing the peak area of phosphorylated abltide (1417 Da) with the peak area of 30 μM abltide from LC (1336.6 Da). Triplicate experiments were conducted on three independent days.

Synthetic peptides (50 μM final concentration) were incubated with 40 units of recombinant human Cdk1-cyclin B (New England Biolabs) in 1× NEBuffer for Protein Kinases (New England Biolabs) with 100 μM cold ATP and 5 μCi γ-[³²P]ATP in a reaction volume of 20 μl. Reactions were incubated at 30°C for 30 min, then terminated by the addition of 10 μl 7.5 M guanidine-hydrochloride. 1.2 μl of each reaction was spotted onto an avidin-coated membrane (SAM² biotin capture membrane, Promega). After air-drying, the membrane was rinsed once with 2 M NaCl, then incubated for 3 × 2 min in 2 M NaCl, 4 × 2 min in 2 M NaCl + 1% H₃PO₄, rinsed twice in distilled water, and air-dried at room temperature for 1 hr. The dried membrane was exposed to an autoradiography film (Carestream BioMax MR) overnight at −80°C.

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.

Peptides 19 amino acids long were synthesized on cellulose membranes using SPOT technology^{48 (link)}. Membranes were rinsed with 95% ethanol, followed by washing 5× with TBST. GSK3 kinase (NEB) was then prepared in a reaction mixture comprising 50 ng of kinase, 200 μM ATP (Sigma-Aldrich) and 8 µCi [γ-³²P]-ATP (Perkin Elmer) in 1.5 ml of 1× NEBuffer for Protein Kinases (NEB). The reaction mixture was then placed on the membrane, which was incubated at 30 °C for 30 min. The membrane was then washed 15× with NaCl (1 M), followed by washing 10× with water before washing with stripping buffer (1% SDS, 8 M urea, 0.5% 2-mercaptoethanol) for 1 h at 40 °C. This was followed by washing 10× in water and a final wash in 95% ethanol. After air drying the membrane for 10 min, it was exposed to a phosphor screen overnight for 3 h. Images were acquired with a PMI phosphorimager (Bio-Rad).