Amersham protran 0.2 nc

Fractionation of nuclear and cytoplasmic fractions was done with the NE-PER kit (Thermo Scientific). Protein samples were separated by 15% SDS-PAGE and then electroblotted onto nitrocellulose membranes (Amersham Protran 0.2 NC, GE Healthcare), blocked with 5% non-fat dry milk in Tris-buffered saline +0.1% Tween (TBS-T). Probing of the blots was done with monoclonal anti-FLAG (F7425, Sigma) in TBS-T and 5% non-fat dry milk, or anti-DDX5 (D15E10, Cell Signaling) in TBS-T, washed three times in TBS-T and 5% non-fat dry milk, followed by incubation with horseradish-peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Sigma) for 2 hr in TBS-T and 5% non-fat dry milk. Protein signals were detected with chemiluminescence imaging (Amersham Imager 600RGB).

An in vitro kinase assay using radiolabeled ATP was performed in 10 mM Hepes, pH 7.6, 50 mM KCl, 5 mM MgCl₂, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, and protease inhibitor (Fig. 3 A). E. coli BL21(DE3) pLysS expressing WT or the S96A mutant version of GST-Ncd(58–192) were sonicated in the kinase buffer and spun down, and then 19 µl of the crude lysate was taken. 1 µl of human PKD2 (14-506; EMD Millipore) diluted in kinase buffer to ∼50 ng/µl or 1 µl of human CaMKIIα (BML-SE470) diluted in kinase buffer to ∼50 ng/µl was added. Approximately 5 µCi of [³²P]γ-ATP was added for 1 h at room temperature, and the sample was boiled in protein sample buffer. The same approach was used for the nonradiolabeled in vitro phosphorylation except that 200 µM of unlabeled ATP was used (Fig. 3 B). For Western blots, proteins were transferred onto nitrocellulose membranes (Amersham Protran 0.2 NC; GE Healthcare) and generally stained for total protein using a Reversible Protein Stain kit for Nitrocellulose Membranes (Thermo Fisher Scientific).