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Fig. 1. Pathogenic mycobacteria express a 50 kDa coronin-1-interacting protein. (A) Macrophages were exposed to radiolabeled live (L) or gentamycin-killed (GK) BCG or live M. smegmatis (L-Msmeg) for 1 hour at 37°C. Partially attached, non-ingested bacteria were removed by a 5 minute treatment with trypsin-EDTA and extensive washing with HBSS and cells were replenished with culture medium and cultured at 37°C. At 4 hours post-phagocytosis cell lysates were prepared and soluble proteins were mixed with anti-coronin-1 mAb (N-7) or normal mouse IgG (Irr). (B) Cell lysates were prepared as described in A, and then mixed with increasing amounts of purified GST-coronin-1 prior to the addition of N-7 mAb. (C) ³⁵S-CFPs from M. smegmatis, BCG and Mtb were incubated with the soluble fraction of J774 lysates and then mixed with N-7 mAb. Material attached to the N-7 mAb was then immunoprecipitated with protein A-agarose and protein complexes were subjected to SDS-PAGE and autoradiography as described in Materials and Methods (A-C). To ensure that coronin-1 levels remained equivalent at the end of immunoprecipitation process, during the last wash of the immunoprecipitates, 10% from each treatment sample was collected and analyzed by SDS-PAGE and immunoblotting with rabbit polyclonal anti-coronin-1 and the secondary goat anti-rabbit IgG (lower panel). (D) Pulled-down CIP50 from BCG ³⁵S-CFPs was solubilized in 2D-gel rehydration solution, and analyzed by 2D-gel electrophoresis and autoradiography.

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