Fig. 2. Cholesterol stabilizes the binding of CIP50 to coronin-1. (A) Aliquots of ³⁵S-CFPs were mixed with agarose-immobilized GST-tagged coronin-1 (or GST alone), in the absence or in the presence of native or denatured coronin-1-depleted soluble J774 proteins (cell lysate), or in the presence of 1 µM cholesterol (water soluble MCD-cholesterol complex) or 10 µM GM1 or 10 mM MCD. The mixtures were then incubated for 2 hours at 4°C and the agarose beads were washed extensively. J774 lysates were depleted of coronin-1 by multiple cycles of immunoprecipitation with N-7 mAb. To ensure that GST-coronin-1 levels remained equivalent at the end of immunoprecipitation process, during the last wash of the immunoprecipitates, 5% from each treatment sample was collected and analyzed by SDS-PAGE and immunoblotting with anti-GST mAb and the secondary goat anti-mouse IgG (lower panel). (B) J774 cells were infected with radiolabeled BCG for 1 hour as described in Fig. 1 then 10 mM MCD was added to the culture plate for additional 30 minutes. After extensive washing of the cells with HBSS, the culture medium was replenished with or without 1 µM cholesterol, and the cells cultured at 37°C for an additional 4 hours. Cell lysates were then prepared and subjected to immunoprecipitation with N-7 mAb. Pulled-down complexes shown in A and B were analyzed by SDS-PAGE and autoradiography. Lower panel in B shows coronin-1 western blotting of 10% immunoprecipitate from each treatment sample. Band intensities were determined by densitometry using the ImageJ software (http://rsb.info.nih.gov/ij).