γ 32p atp

Recombinant viral proteins P1, P2, P3, P6/TAV and GST-P1P2(D2) were expressed in Escherichia coli BL21(DE3)pLysS transformed with the respective plasmids (see “Plasmids” section for details concerning their construction). Their expression was induced in bacteria culture in exponential phase by adding 1mM Isopropyl β-D-1-thiogalactopyranoside. Bacteria were then grown at 28°C for 4 h, pelleted and suspended in heart muscle kinase (HMK) buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 12 mM MgCl₂). After being sonicated 3 times for 10 s, lysates were centrifuged at 11,000 X g, for 10 min at 4°C and the inclusions bodies were suspended in 0.5 mL HMK buffer. 30 μl of the inclusion bodies suspension were phosphorylated in the presence of 25 μCi [γ-³²P]ATP and 25 U of HMK (Sigma-Aldrich) in 150 μl HMK buffer, at room temperature for 1.5 h. ATP in excess was eliminated using an illustra MicroSpin G-25 Column (GE Healthcare).

Protein lysates were prepared as described by Hlavová et al. [32 (link)]. They were directly assayed or affinity-purified by CrCKS1 beads as described by Bisova et al. [19 (link)] with modifications as described by Hlavová et al. [32 (link)]. Histone H1 kinase activity was assayed as previously described [33 (link)] in a final volume of 10 µL with either 7 µL of clear whole cell lysate or the CrCKS1 beads fraction corresponding to 20 µL of whole cell lysate. The reactions were initiated by adding the master mix to a final composition of 20 mM HEPES, pH 7.5, 15 mM MgCl₂, 5 mM EGTA, 1 mM DTT, 0.1 mM ATP, 0.2% (w/v) histone (Sigma H5505) and 0.370 MBq [γ ³²P] ATP. All the chemicals were purchased from Sigma-Aldrich (Prague, Czech Republic).
Proteins were separated on 15% SDS-PAGE gels [34 (link)]. Phosphorylated histone bands were visualized by autoradiography, analyzed using a Phosphoimager (Storm 860, Molecular Dynamics, GE Healthcare, Prague, Czech Republic), and quantified using Image Studio Lite software (LI-COR Biosciences, v. 5.2, Lincoln, NE, USA) as described by Zachleder et al. [4 (link)].

Neurons were lysed in ice-cold buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% NP-40, supplemented with the phosphatase and protease inhibitors cited above. After clearing debris by centrifugation, extracts (200 μg protein) were incubated with anti-Cdk2 (1 μg) or anti-Cdk4 (1 μg) for 4 h at 4 °C, followed by the addition of 30 μl of protein A-sepharose (GE Healthcare Life Sciences) for 2 h at 4 °C. Immunoprecipitates were washed four times in lysis buffer and resuspended in kinase buffer (50 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EDTA and 0.1 mM dithiothreitol) containing 20 μM ATP, 2 μCi of [γ-32P]ATP and either histone H1 (1 mg/ml; Sigma), for Cdk2, or pRb (1 mg/ml; Calbiochem), for Cdk4. Samples were subjected to SDS-polyacrylamide gel (12%) electrophoresis and transferred proteins were visualized by autoradiography or blotted with either anti-Cdk2 or anti-Cdk4.

[³²P]PEP was prepared enzymatically as described previously in ref. 48 (link). Briefly, 50 μl reaction solution containing 100 mM triethylamine/HCl pH 7.6, 15 mM KCl, 3 mM MgCl₂, 165 μM PEP, 1 mM pyruvate, 5 μM ATP, 60 μCi [γ-³²P]-ATP (5,000 Ci mmol⁻¹) and 40 units of pyruvate kinase (Sigma) were incubated at 30 °C for 2 h. Phosphorylation assays were performed in 20 μl of solution containing 10 μl of proteins extract (containing EI^Ntr or EI^Ntr_L83Q), 25 mM Tris/HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, glutamine (0, 2, 5 or 10 mM) and 0.5 μl of [³²P]PEP solution at 37 °C for 30 min. Then, 5 μl of 5 × SDS-PAGE loading buffer were added to the samples. Proteins were subjected to electrophoresis in a 10% SDS-polyacrylamide gel. SDS-polyacrylamide gels were then dried and imaged on a MP Phosphor system (Packard) and analysed with Cyclone Phosphor Imager (PerkinElmer). Analysis of radioactive spots reveals three bands at different size (∼50, 80 and 90 kDa). The band corresponding to ∼80 kDa, absent in protein extracts from the ΔptsP strain, was determined as EI^Ntr or EI^Ntr_L83Q.

Cell extracts from a nearly confluent T25 flask were lysed in buffer (see above) and incubated with 1 µg of either anti-CDK2 antibody (BD Biosciences, 610145) or anti-GFP antibody (DSHB, 12A6; used as a negative control) overnight. Antibody–antigen complexes were precipitated using protein A and G sepharose beads (GE Healthcare). Bead complexes were washed three times with lysis buffer. Kinase assay reactions were incubated at 30 °C for 30 min in buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl₂, 0.1 mM NaF, 10 μM Na₃VO₄) with 1 mM DTT, 1 μM cold ATP, 2 μCi [γ-³²P] ATP, and 4 μg histone H1 (Sigma, H1917). Reactions were resolved by SDS-PAGE. Dried gels were exposed to a storage phosphor screen (GE Healthcare), and γ-³²P incorporation was visualized by Typhoon TRIO imager (GE Healthcare).