Fig. 7.

Effects of SM acyl chain length on VacA distribution in rafts.

AZ-521 cells were incubated with SMase C (50 mU/mL) for 1 h at 37 °C, and then chillled on ice to 0 °C. The cells were washed with ice-cold-medium to remove SMase C. The cells were then incubated in the presence of C2-SM, C12-SM, or C18-SM (25 μM) for 30 min on ice. The cells were further incubated with prechilled VacA (100 nM) for 1 h on ice, washed once with ice-cold medium, and lysed and analyzed for raft and non-raft proteins as described under Experimental Procedures.

(A) Western blot analysis of VacA and flotillin in DRM and non-DRM fractions of lysed cells.

(B) Fraction of VacA in DRM and non-DRM fractions of lysed cells as determined by densitometry analysis of Western blots. These results are rendered from the combined data obtained from three independent experiments. Statistical significance was calculated for differences between the cross-reacting material present in the DRMs and non-DRMs.

(C) Western blots analysis of VacA and actin cross-reacting material from total lysates of cells enriched in C2-SM, C12-SM, C18-SM, or mock-pretreated cells.