Typhoon trio imaging system

E12.5 telencephalic lobe extracts were incubated ON at 4°C with saturating concentrations of Cyclin E, Cdk2, or Cyclin D1 Abs. Immune complexes were collected by incubation with Dynabeads Protein G (invitrogen) for 1 h at 4 °C, and equilibrated on kinase buffer (50 mM TrisHCl pH7.5, 20 mM EGTA, 10 mM MgCl₂, 1 mM DTT, 1 mM β-glycerolphosphate). For the kinase assays, 10 ml of kinase reaction mixture (0.4 mM ATP pH 7.5, and 10 mCi [γ-^{32 (link)} P]ATP (Amersham Pharmacia)) in kinase buffer containing substrate (2.5 µg of histone-H1 (Sigma-Aldrich) for Cyclin E and Cdk2, and 0.2 µg of soluble GST-pRb fusion protein (Santa Cruz Biotechnology) for Cyclin D1, was added to the immunoprecipitated complexes. After 30 min of incubation at 37 °C, reactions were stopped by adding 5 µl of Laemmli loading SDS buffer and heating at 100 °C for 10 min. Labelled proteins were resolved by 15% SDS-PAGE. Gels were incubated twice in 20% methanol; 10% glycerol solution before dried on 3 mm paper at 50 °C with vacuum. Images were acquired on Typhoon TRIO + imaging system (Amersham). For Abs and dilutions, see Supplementary Table 6.