Fig 1.

CR3-mediated phagocytosis of B. burgdorferi requires the translocation of the integrin to lipid rafts. (A) RAW264.7 cells were incubated for 1 h with B. burgdorferi (m.o.i. = 25), lysed and layered onto a sucrose gradient. The gradients were separated by ultracentrifugation for 20 h. Ten fractions were immunoblotted with anti-CD11b and anti-CD14 Abs. Fraction numbers from the top are indicated. T: whole lysates (25 μg). (B) RAW cells were incubated with Bb914 for 1 h in the presence of CTxB for the last 20 min. The cells were fixed and images obtained by confocal microscopy. The images represent orthogonal projections showing colozalization or lack thereof between Bb914 (green) and CTxB (red). (C) Confocal micrographs showing CD11b localization to lipid rafts. RAW264.7 cells were incubated with Bb914 (lower panels) or left unstimulated (upper panels) for 30 min. The cells were washed, fixed and stained with an anti-CD11b Ab (blue) and CTxB (red). Colocalization of CD11b and CTxB staining is indicated by the purple color in the small panel. The results presented are representative of 3-5 individual experiments.