Monoclonal antibody probes for p21(WAF1/CIP1) and the INK4 family of cyclin-dependent kinase inhibitors

Inhibition of cyclin dependent kinases (cdk) by proteins of two families of cdk inhibitors (CKIs) represents one of the key modes of cell-cycle contro l. Although not fully understood at present, the functions of the individua l members of the Cip/Kip and INK4 families of CKIs have been implicated in fundamental biological processes as diverse as cellular proliferation, resp onses to genotoxic stress, regulation of cellular differentiation, and sene scence. In addition, the seven currently known CKIs qualify as either estab lished or candidate tumor suppressors whose loss or inactivation contribute to molecular pathogenesis of a wide range of tumor types. In this study, w e report the isolation and characterization of a panel of 10 mouse monoclon al antibodies (MAbs) that specifically recognize p21(WAF1/CIP1) (p21) or th e individual members of the INK4 family of CKIs: p15(INK4b) (p15), p16(INK4 a) (p16), p18(INK4c) (p18), or p19(INK4d) (p19). These antibodies are provi ng to be invaluable molecular probes for analyses of protein abundance, sub cellular localization, interacting cellular proteins, and ultimately the fu nction(s) of these cell cycle regulators. Epitopes targeted by the antibodi es were mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and performance of the MAbs assessed in a range of immunochemical techniques. I ndividual MAbs of our series recognize distinct pools of the respective CKI s, a feature reflected by their differential applicability in immunoblottin g, immunoprecipitation, and immunostaining including immunohistochemistry o n archival paraffin-embedded tissue sections. Together, these antibodies re present useful reagents to study CKIs in cells and tissues, a set of tools that should help elucidate the physiological roles played by the individual CKIs, and better understand the molecular mechanisms of loss or inactivati on of these (candidate) tumor suppressors in human malignancies.