2.10. Kinase Assay

U1 cells (5 × 10⁶) were treated with CBD (1 µM) and THC (3.18 µM) every day for 5 days. Cells were pelleted by centrifugation at 15,000× g for 5 min, washed with 1× PBS, and lysed in 250 µL lysis buffer. Lysates were immunoprecipitated with 10 µg of cdk9 or IgG antibodies overnight at 4 °C, followed by a 2 h incubation with A/G beads at 4 °C. IP complexes were washed with TNE50 + 0.1% NP40 buffer and kinase buffer as described in [27], followed by 1 h incubation period at 37 °C with γ-³²P ATP and purified histone H1. The samples (50%) were loaded and run through SDS-PAGE on a 4–20% Tris-Glycine gel, followed by staining with Coomassie blue overnight and then destained with destaining buffer (50% methanol and 10% acetic acid), and dried for 2 h. The dried gels were exposed to a PhosphorImager Cassette, followed by analysis using Molecular Dynamic’s ImageQuant Software; Los Altos, CA, USA.