Figure 2.

Cytotoxicity of recombinant EcPltAB. 18 h after treatment with buffer or different concentrations of EcPltAB, Vero cells were stained and imaged. a, representative images for untreated controls (left) and 50 ng/ml EcPltAB (right) are shown. b, ADP-ribosyltransferase assays in which pertussis toxin or EcPltAB were used to modify different protein substrates, including trimeric G-protein extracts, recombinant human Gα_i3, or Vero cell lysates. Reactions at 37 °C were performed in the presence of biotin-NAD⁺ and then quenched after 5 min, separated by SDS-PAGE, transferred to a Western blotting membrane, and imaged with either luminescence following streptactin-HRP exposure (left) or Coomassie (right). c, Western blottings showing the incubation of ExPEC culture medium with NAD⁺-biotin with and without the presence of HsGα_i3ΔN. d, Vero cells were imaged in a similar manner to those in a were monitored for the degree of cell growth relative to buffer controls following exposure to different concentrations of EcPltAB or A subunit mutants. Surface area is calculated from the percentage of each image stained by crystal violet. Data represent the mean ± S.D. from three replicates.