Specific monoclonal antibodies to the p!5(INK4b) and p16(INK4a) cyclin/cdk inhibitors

Cyclin-dependent kinases (cdks) are activated by the binding of cyclin proteins and.phosphorylation events. Timing the appearance of specific cyclins is one way in which the cell regulates key events in the progression of the cell cycle. Inhibitors of cyclin/cdk complexes provide another level of regulation on the cdk activity and, as such, are important for their effects on the cell cycle and for their potential involvement in tumor suppression. p!6 was the first member of a growing family of inhibitors specific for cdk4 and/or cdko. The family now includes p!5(INK4b), p!6(INK4a), p!8(INK4c), and p!9(INK4d), all of which share significant sequence homology. Mutations in pi 6 have been found in several tumor types and appear most often in tumors which carry an intact Retinoblastoma protein, pi 5 is involved in the cellular growth arrest induced by TGF-beta. We have developed several mAbs against the human p!5 and p!6 cdk inhibitors which are useful in investigating cell cycle regulation. Both p!5 and p!6 antibodies were purified from hybridomas made from mice immunized with GST-pl5 or GST-pl6. The hybridomas were initially screened by ELISA against the corresponding recombinant proteins. Hybridomas were also screened for reactivity to control GST fusion proteins to eliminate clones with unwanted reactivity. Further selection for reactivity in western blot identified clones which specifically reacted with recombinant forms of either p!5 or p!6 or both. Clones were tested for utility in immunoblotting, immunoprecipitation and immunocytochemistry.