The hNIAM protein was quantified and used for screening the monoclonal antibodies. Immunization of mice and generation of hybridomas Two female Balb/c mice (National Cancer Institute, Frederick, MD) were immunized with three rounds of injections per animal using 100 transcription and translation (TNT kit, Promega, Madison, WI) using 35S-TransLabel (ICN). gene or deregulation of its many activators and inhibitors, removes those protective brakes to the cell cycle and is a defining feature of nearly all human cancers.(3) Indeed, genetic inactivation of occurs in over 50% of human tumors(4) while loss of its key activator, the alternative reading frame (ARF) tumor suppressor,(5) is the second most common event during carcinogenesis.(6) Most human cancers also overexpress (R)-Nedisertib the p53 antagonist Mdm2, which likewise results in p53 inactivation.(7C9) ARF stimulates p53 in response to aberrant oncogenic signaling and is essential for maintaining its activity following DNA damage.(7,10) Most evidence suggests that ARF primarily functions (R)-Nedisertib by binding and inhibiting Mdm2, an E3 ubiquitin ligase that targets p53 for degradation.(8) However, ARF has many known binding partners and can prevent cancer independent of p53 through antiproliferative pathways that are only partly defined.(8,11) We recently discovered several new binding partners of ARF that contribute to both its p53-dependent and p53-independent signaling pathways(12,13) (also, unpublished data, V. Tompkins and D.E. Quelle). One of those partners is a novel protein we named because it was found to be a nuclear interactor of ARF and Mdm2.(13) NIAM is normally expressed at low levels in cells due to Mdm2-mediated ubiquitination and degradation. When overexpressed, (R)-Nedisertib NIAM inhibits cell cycle progression, enhances ARF stability, and activates p53. NIAM also has undefined ARF-and p53-independent activities that help it maintain chromosomal stability. Little else is currently known about the normal function and regulation of NIAM during tumorigenesis, although the above data strongly suggest NIAM may be a tumor suppressor protein. A major impediment to studying NIAM’s role in cancer, however, has been the inability of existing NIAM polyclonal antibodies to detect endogenous NIAM protein expression in normal and transformed human cells. Therefore, we began the development and characterization of monoclonal antibodies (MAbs) to human NIAM. Here we describe the identification of several MAbs that effectively recognize endogenous human NIAM protein using multiple molecular approaches. Materials and Methods Bacterial protein expression and purification Wild-type human NIAM (hNIAM) cDNA was subcloned into the pGEX-2T vector and expressed as a glutathione S-transferase (GST) fusion protein in BL21 following induction with IPTG (1 mM) for 3 h at 37C. Soluble GST-hNIAM protein was recovered from bacterial cell lysates on glutathione S-Sepharose (Amersham Biosciences, Piscataway, NJ), washed three times in NETN lysis buffer (120 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl [pH 8.0], 0.5% NP-40), and eluted with 20 mM glutathione in elution buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, 5 mM (R)-Nedisertib DTT [pH 8.0]). GST-hNIAM was then dialysed into phosphate-buffered saline (PBS) and concentrated to approximately 1 mg/mL using centricon-30 filtration units (Millipore, Bedford, MA), as described by the manufacturer. Purified GST-hNIAM was quantified by BCA assay (Pierce Biotechnology, Rockford, IL) and used as antigen to immunize mice. Untagged hNIAM protein was then recovered from the remaining Sepharose-bound GST-hNIAM pool by cleavage with thrombin (Amersham Biosciences) and separated from GST Rabbit polyclonal to HOXA1 by SDS-PAGE, and the band containing the protein was sliced out of an unfixed Coomassie-stained gel (0.05% Coomassie blue in ddH2O for 5 min). The protein was extracted from the gel by overnight incubation at 30C in 2.5% 2-mercaptoethanol, 1% SDS, and 50 mM Tris-HCl [pH 6.8]. The majority of SDS was removed by precipitation at 4C and the sample was concentrated and dialysed into PBS as described above. The hNIAM protein was quantified and used for screening the monoclonal antibodies. Immunization of mice and generation of hybridomas Two female Balb/c mice (National Cancer Institute, Frederick, MD) were immunized with three rounds of injections per animal using 100 transcription and translation (TNT kit, Promega, Madison, WI) using 35S-TransLabel (ICN). Labeled proteins were then immunoprecipitated by overnight incubation at 4C with each MAb supernatant (100 translated (IVT) hNIAM (Fig. 3). Immune complexes were captured on Protein G-agarose, separated by SDS-PAGE, and the presence of immunoprecipitated hNIAM detected by autoradiography. The most effective MAbs able to IP hNIAM were I-G5, VIII-E101, I-G21, and VII-C82 (Fig. 3). By comparison, only I-G5 recognized a mNIAM IVT product by IP (data not shown). Other MAbs either failed to effectively IP hNIAM (I-G22, V-E43, and VIII-H3) or did so at an intermediate (R)-Nedisertib level (VII-C81 and VIII-E102). These results, as well as data from subsequent assays described below, are summarized in Table 1. 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