FIGURE 5.

Localization of Ral in PMN. A, Ral is associated with membranes. PMN (2.5 × 10⁷) were Dounce-lysed, followed by ultracentrifugation to separate cytosol from cell membranes. SDS-PAGE and Western blot were performed to detect Ral in the total lysate before centrifugation (total PMN) and in the separated cytosol and membranes. B, subcellular localization of Ral in PMN. PMN (10⁸/condition), unstimulated and fMLF-stimulated, were lysed and subjected to sucrose density gradient centrifugation to separate the plasma membrane and intracellular granules, followed by fractionation. Western blots (lower panel) were performed to detect Ral, SIRPα, and JAM-A in the fractions using antibodies against Ral, anti-SIRPα.ex, and anti-JAM-A.ex. JAM-A is a transmembrane protein consistently expressed in the plasma membranes.