Figure 4. Exogenous SM Rescues VacA Binding to SM-Depleted Cells.

Monolayers of HeLa cells were pretreated with SMase C (100 mU/mL) or mock-pretreated with PBS pH 7.2 at 37 °C and under 5% CO₂. After 1 h, the cells were washed, chilled to 0 °C on ice, and incubated for 1 h with exogenous SM at the indicated concentrations. The cells were then incubated with Alexa Fluor 488-labeled VacA (10 nM) or lysenin-GFP (1 μM) at 4 °C. After 1 h, protein binding was quantified by flow cytometry. Cell binding was normalized to the mean geometric fluorescence of Alexa Fluor 488-labeled VacA binding to mock-pretreated cells. Asterisks indicate statistically significant differences in VacA binding to cells treated with SMase C and mock-pretreated cells.