Myelin basic protein

Ninety-six well-polystyrene plates (Thermo Scientific™ Immuno non-sterile 96-well Nunc MaxiSorp™ flat-bottom) were coated for 1 h at room temperature with 2 μg/ml of Factor VIII (Kogenate FS, Bayer, Munch, Germany), with 10 μg/ml of Factor IX (LFB Biomedicaments, Les Ulis, France), Histone 3 (Sigma-Aldrich), or Myelin basic protein (Sigma-Aldrich) in PBS buffer. Plates were blocked with 0.25% Tween 20 in PBS for 90 min. After the plates were incubated 100 μg/ml of pooled IgG preparations for 2 h at room temperature. After washing with PBS containing 0.05% Tween 20, mouse anti-human IgG antibody conjugated HRP (Southern Biotech, Birmingham AL, USA) was added and incubated for 1 h at room temperature. After series of washing with PBS containing 0.05% Tween 20, immunoreactivities were revealed by adding o-phenylenediamine dihydrochloride (Sigma-Aldrich) diluted in phosphate citrate buffer pH 5. The absorbance values were read at 492 nm after stopping of the reaction with 2N HCl.

Aurora A kinase activity was assayed by measuring incorporation of γ³³P-ATP into myelin basic protein (Sigma). Purified recombinant protein (100 ng) was incubated at room temperature for 30 min in a total volume of 15 μl containing 50 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.05% Brij-35, 0.5 mg/ml myelin basic protein, and 5 μM γ³³P-ATP (100–10,000 dpm/pmol). The reaction was stopped by spotting the reaction mixture onto squares of P81 phosphocellulose ion exchange paper (Whatmann), which were then immersed in 1.5% (v/v) phosphoric acid. After washing twice in 1% phosphoric acid followed by two washes in distilled water, the papers were air dried and radioactivity was counted by a scintillation counter and recorded by Quantasmart version 2.03.

Kinases were provided as recombinant proteins purified from E. coli or immunoprecipitates from mitotic cell lysates. For IP-kinase assays, the immunoprecipitates were washed twice with cell lysis buffer (1× PBS, 10% glycerol, 0.5% NP-40) supplemented with protease inhibitors (Protease Inhibitor Cocktail set III, EDTA-Free; Calbiochem) and phosphatase inhibitors (100 mM NaF, 1mM Na₃VO₄, 60 mM β-glycerophosphate) and twice with 1× kinase buffer (25 mM Tris-HCl, pH 7.5, 60mM ß-glycerophosphate, 10mM MgCl₂). Myelin basic protein was purchased from Sigma and Histones H3.3 and H1₀ were purchased from New England Labs as substrates. For kinase reactions, 4μl of 5× kinase buffer was mixed with recombinant or immunoprecipitated kinases, substrates, 5μCi ³²P –ATP or cold ATP. H₂O was added to make the final volume of 20 μl. The reactions were incubated at 30°C for 30 min and then terminated by adding 20 μl 2×SDS sample buffer. Samples were subjected to SDS-PAGE followed by transferring to PVDF membranes (Millipore). Phosphorylation of the substrates was visualized by autoradiography or phospho-specific antibodies.

HEK293T cells were transfected with HA-PRMT5 expression plasmids and immunoprecipitated with an anti-HA antibody. A recombinant substrate (5 μg) either GST-GLI1 protein or myelin basic protein (Sigma-Aldrich) as a positive control for a PRMT5 substrate, was incubated with immunoprecipitated HA-PRMT5. S-adenosyl-l-methyl-[³H] methionine (55 Ci mmol⁻¹; Perkin Elmer) was added to each reaction in a total volume of 40 μl MRE, and samples were incubated for 1 h at 30 °C. Samples were separated by SDS–PAGE, and the gel was washed and incubated in amplify solution (Perkin Elmer). The gel was then dried and exposed on BIO-MAX MR X-ray film (Carestream) for 3–7 days at −80 °C.

The procedure was performed as described in Mazur et al. (2021) (link). In brief, for immunoprecipitation of GFP-fused proteins, 400 µg of crude protein extract from seedlings treated with H₂O₂ or MV was incubated with 10 µL of GFP-Trap^®_A (Chromotek) for 2.5 h with gentle rocking. After intensive washing, agarose beads with bound immunocomplexes were suspended in 20 mM Tris–HCl, pH 7.5 supplemented with 150 mM NaCl and 4 µg of Myelin Basic Protein (Sigma-Aldrich) per sample. To each sample, ATP supplemented with 1 μCi of [γ³²P]ATP in kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM EGTA, 1 mM DTT, 30 mM MgCl₂) was added to 50 μM final concentration. After 15 min of incubation at 37°C samples were mixed with Laemmli sample buffer and incubated for 3 min at 95°C with vigorous shaking. Proteins were separated on 12% SDS polyacrylamide gel and signal was detected on Medical X-ray Blue/MXBE Film (Carestream).

Coding sequences for GFP, S6KL, and S6KL^K193Q were subcloned into pAC5.1 plasmids to produce N-terminally Flag tagged fusion proteins. Recombinant plasmids were transfected into S2 cells by Cellfectin II reagent (Invitrogen). After 48 h, transfected cells were lysed in RIPA buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP-40) for 1 h at 4°C. Cell lysates were precleared with protein G-Sepharose (GE) for 1 h and then incubated with anti-Flag monoclonal antibody (Sigma) for 3 h at 4°C. The immuno-complexes were precipitated with protein G-Sepharose (GE), washed five times with RIPA buffer, and then washed two times with kinase buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM β-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na₃VO₄, and 10 mM MgCl₂. To perform the kinase activity assay, the anti-Flag immunoprecipitates were incubated with 50 μM ATP (Sigma), 10 μCi [γ-³²P] ATP (PerkinElmer) and 5 μg myelin basic protein (Sigma) as substrates for 40 min at 30°C. The reaction products were separated on 12% SDS-polyacrylamide gels and autoradiographed to detect γ-³²P incorporation.

In-gel kinase assays for replication checkpoint activation were conducted as described previously (Geahlen et al, 1986 (link); Waddell et al, 1995 (link); Takeda et al, 2001 (link)). SDS–polyacrylamide gel (10%) was cast in the presence of 0.5 mg/ml myelin basic protein (Sigma-Aldrich) within the gel. Extracts (100 µg of protein) prepared by the boiling method were run on the gel. After electrophoresis, the gel was washed successively in 50 mM Tris–HCl (pH 8.0), 50 mM Tris–HCl (pH 8.0) +5 mM 2-mercaptoethanol, and denatured in 6 M guanidium hydrochloride (Nacalai tesque) in 50 mM Tris–HCl (pH 8.0) +5 mM 2-mercaptoethanol, and renatured in 50 mM Tris, pH 8.0 +5 mM 2-mercaptoethanol +0.04% Tween 20 over 12–18 h at 4°C. The gel was then equilibrated in the kinase buffer containing 40 mM HEPES-KOH (pH 7.6), 40 mM potassium glutamate, 5 mM magnesium acetate, 2 mM dithiothreitol, and 0.1 mM EGTA for 1 h at room temperature, and was incubated in the same kinase buffer containing 5 µM ATP and 50 µCi of [γ-³²P]ATP for 60 min at room temperature, followed by extensive washing in 5% trichloro-acetic acid (Nacalai tesque) +1% sodium pyrophosphate until no radioactivity is detected in the washing buffer. The gel was dried and auto-radiographed.