Fig. 6. Intracellular expression of LRG-47 dislocates coronin-1 from mycobacterial phagosomes. (A) Lysates from control and IFN-stimulated J774 cells were examined by SDS-PAGE and immunoblotting with anti-LRG-47 antibodies, then membranes were stripped and reprobed with anti-actin antibodies. (B) J774 cells were transfected with GFP or GFP-LRG-47 as described in Materials and Methods. In a first series of experiments (a,b), cells were preloaded with LysoTracker Red and infected with BCG expressing Ebfp (BFP-BCG) then fixed at 4 hours post-phagocytosis. In a second series of experiments (c and d), transfected cells were directly infected with BFP-BCG and stained with coronin-1 at 4 hours post-phagocytosis. In b, the white signal indicates simultaneous colocalization of BCG vacuoles with LysoTraker and LRG-47. In c, the magenta signal indicates colocalization of BCG with coronin-1. The cyan signal in d indicates colocalization of BCG phagosome with LRG-47. Short arrows indicate the position of BCG bacilli. (C) Quantification of BCG-LysoTracker and BCG-coronin-1 colocalizations in phagosomes observed in 15-20 cells from two individual experiments. (D) Upper panel: control, IFN-stimulated, GFP- or GFP-LRG-47-transfected cells were infected with radiolabeled BCG and at 4 hours post-phagocytosis, intracellular CIP50-coronin-1 interaction was examined as described in Fig. 1. The relative amounts of CIP50 co-immunoprecipitated with coronin-1 were determined by radioactive count of small aliquots of immunoprecipitated material. Lower panel: total cell lysate form the same treatments were also examined for expression of coronin-1 by western blotting with anti-coronin-1 antibodies.