5a). recognition by SW5 reside in elements adopting alpha-helical conformations. These data indicate that the application of specific mAbs to purify and characterize (functionally) interacting proteins can be severely obscured by the cross-reactivity of mAbs with structurally, but not functionally, similar proteins. Introduction The human La (SS-B) protein is an RNA-binding phosphoprotein that is frequently targeted by autoantibodies occurring in sera from patients with diseases like systemic lupus erythematosus and Sj?gren’s syndrome.1,2 This abundant protein has been identified in many eukaryotic organisms including and for 1 hr. Constructs and recombinant protein expressionFor transcription/translation an N-terminally vesicular stomatitis virus glycoprotein epitope (VSV-G)-tagged La protein construct was used.14 Recombinant human La was expressed and purified as described previously.15 An N-terminally VSV-G-tagged human EEA2 construct has ON 146040 been described (Fouraux translated 35S-methionine labelled wild-type EEA2 or La protein were synthesized using the TNT T7 Quick Coupled Reticulocyte Lysate System (Promega, Leiden, the Netherlands), using the VSV-G-tagged versions of EEA2 and La in the pCI-neo vector as templates. For immunoprecipitation analysis, protein A-agarose beads were coated with SW5 or SW3 as described above. 35S-methionine labelled EEA2 or La proteins were incubated with the mAb-coated beads in IPP300 (10 mm TrisCHCl, pH 80, 300 mm NaCl, 005% NP-40) for 1 hr at 4. After extensive washing, bound protein was eluted in SDS-sample buffer, separated by SDSCPAGE and visualized by autoradiography. For competition assays, the mAb-coated beads were preincubated with increasing amounts of recombinant La (0, 01, 1 and 10 g recLa) prior to the incubation with 35S-methionine labelled EEA2 or La proteins. Results SW5 precipitates EEA2 from a HeLa S100 extract To identify proteins interacting with La we performed preparative immunoprecipitations from HeLa S100 extracts with the mAb SW5. Co-precipitated proteins were eluted from SW5-coated protein A-agarose beads with 1 m NaCl and were fractionated by 12% SDSCPAGE. Several proteins were specifically isolated from the extract in addition to the expected La and Ro60 proteins and one of these was identified as EEA2 (Fouraux translated, 35S-labelled EEA2 to immunoprecipitation with SW5. As shown in Fig. 3(a), lane 2, SW5 indeed immunoprecipitated EEA2 independent of the presence of the La protein. Similar immunoprecipitation experiments that were done in the presence of increasing amounts of bacterially expressed recombinant human La protein indicated that La competes for the binding of EEA2 to SW5 (Fig. 3a, lanes 3C5). This result indicates that EEA2 shares antigenic determinants with La, which are recognized by mAb SW5. As expected, under these conditions recombinant La also competed for the binding of translated La (Fig. 3b, lanes 3C5). Open in a separate window Figure 3 Recombinant La competes with translated EEA2 for binding to SW5. Protein A-agarose beads coated with the anti-La mAb SW5 were incubated with increasing concentrations (0, 01, 1 and 10 g recLa) of recombinant La, followed by incubation with translated 35S-methionine-labelled EEA2 (a) or translated 35S-methionine-labelled La (b). Subsequently, the beads were extensively washed, followed by solubilization of the precipitated protein and analysis by 10% SDSCPAGE and autoradiography. Lane 1, translated protein (5% of the amount used in the precipitations); lane 2, SW5 immunoprecipitation; lanes 3C5, SW5 immunoprecipitation in the presence of 01, 1 and 10 g recLa, respectively; lane 6, control precipitation with beads alone and 35S-methionine-labelled EEA2 (a) or 35S-methionine-labelled La (b). SW5 recognizes an epitope in CC3 of EEA2 Since SW5 cross-reacted with recombinant EEA2 protein both on Western blots and ON 146040 in solution, we were interested to delineate the region of EEA2 responsible for the recognition by SW5. To investigate this, we constructed a series ON 146040 of deletion mutants of EEA2 (Fig. 4a). These mutants, as well as wild-type EEA2, were produced by transcription-translation (Fig. 4b, lanes 1C8) and subjected to immunoprecipitation with either SW5 (Fig. 4b, lanes 9C16) or SW3 (Fig. 4b, lanes 18C25). All translated EEA2 mutants were reactive with the anti-EEA2 rabbit serum in immunoprecipitation analyses, suggesting efficient (re)folding of these proteins (data not shown). The results demonstrated that all mutants of EEA2 lacking CC3, namely N-LZ1, N-LZ1-CC1, LZ1-CC12 and LZ1-CC1, were not or only very inefficiently precipitated by SW5 (Fig. 4b, lanes 10, 13, 15 and 16), indicating that CC3 plays an important role in the recognition of EEA2 by SW5. SW3 did not detectably precipitate 35S-labelled EEA2 nor its mutants (Fig. 4b, lanes 18C25), in agreement with the lack of recognition of Mouse monoclonal to FAK EEA2 by SW3. The results of these deletion.
