Binding of Mutant CDT- Alexa Fluor 488 Holotoxin to Cells

<p>HeLa cells were treated for 2 hr with 5 μg/ml concentration of wild-type or mutant fluorescently labeled holotoxin in 3% BSA/PBS at 37 °C, 5% CO₂. Samples were co-stained with Hoechst 33258 dye to visualized nuclei. Toxin was washed out, and cells were fixed in 3.7% formaldehyde and mounted with ProLong Gold Antifade Reagent (Molecular Probes). Confocal images were taken under an inverted LSM510 confocal microscope (Zeiss) using ×60 objective lenses. The aromatic patch mutant contains wild-type CdtB and CdtC, and mutant CdtA (W91G/W98G/W100G/Y102A); the groove mutant contains wild-type CdtB and mutant CdtA (P103A/Y106A), and CdtC (R43K/Q49A).