Ezview anti ha beads

For kinase assays, 1 × 10⁷ cells were lysed in 500 µl of ELB buffer (50 mm HEPES, pH 7.5; 160 mm NaCl; 5 mm EDTA; 0.1% NP-40, 1× protease inhibitors; 1 mm PMSF), and for co-IPs, 5–10 × 10⁶ cells were lysed in RIPA buffer. For IPs, 1 µg of anti-CDK2 or IgG, with 20 µl of Protein A/G agarose (Santa Cruz Biotechnology), or 20 µl of EZview anti-HA beads (Sigma) was used as previously described [9 (link)]. Beads were washed with kinase buffer (25 mm HEPES, pH 7.5; 5 mm MgCl₂; 2.5 mm MnCl₂; 0.5 mm DTT) prior to adding 0.75 µg of recombinant 6 × His-Rb (amino acids 792–928; ProSpec, Ness-Ziona, Israel), 37.5 µm ATP, and 1 μCi of [γ-³³P]ATP for 30 min at 30 °C. Samples were resolved by SDS-PAGE; gels were fixed in 10% acetic acid/methanol, dried, and quantified using a Cyclone Phosphor Imager (PerkinElmer, Waltham, MA, USA).