Figure 1.

Subcellular localization of coronin 1. (A) Macrophages (J774), lymphocytes (Jurkat T-cell lymphoma), or coronin 1-transfected HEK293 cells were grown on microscopy slides, fixed, and permeabilized followed by immunolocalization of coronin 1 and F-actin localization by using anti-coronin 1 antiserum (middle) and Texas Red-labeled phalloidin (bottom). Top, corresponding Nomarski images. Bar, 10 μm. (B) Biochemical analysis of coronin 1 interaction with macrophage membranes. The postnuclear supernatant of a J774 cell homogenate was separated into membrane fraction and cytosol (100,000 × g, 30 min) and analyzed for the presence of coronin 1 by SDS-PAGE and immunoblotting. (C) Biochemical analysis of coronin 1 localization in the detergent-insoluble fraction of macrophages. J774 macrophages were lysed in cytoskeleton isolation buffer containing 1% Triton X-100 and directly subjected to low-speed centrifugation (3000 × g, 2 min; see Materials and Methods). Subsequently, the detergent-insoluble pellets and the supernatants were subjected to SDS-PAGE and immunoblotting for detection of coronin 1, actin, tubulin, vimentin, and the raft marker protein CD14. (D) J774 macrophages were treated for 30 min with 5 μM LatB, processed as in C, and analyzed for the presence of coronin 1, actin, vimentin, and CD14 in pellets and supernatants.