Fig 5.
The CyaAΔAC toxoid delivering OVA257-264 epitope conducts Ca2+ ions into cells and associates with lipid rafts. (A) J774A.1 cells were loaded with the calcium probe Fura-2/AM at a 3 μM final concentration at 25°C for 30 min. After cells were washed in HBSS medium, toxoid variants (17 nM) were added at time zero (arrow), and the time course of calcium entry into the cytosol (elevation of intracellular [Ca2+]) was monitored by spectrofluorimetry (ratiometric measurement) as described elsewhere (10). (B) J774A.1 cells were incubated for 10 min with the indicated toxoids (1 nM). Detergent-resistant membrane microdomains were extracted with cold Triton X-100 and separated by flotation in a sucrose density gradient. The toxoids were detected in gradient fractions with the 9D4 antibody using Western blots. Results of one representative experiment of two are shown.