FIGURE 5.

JKH-C7 does not inhibit preformed PA63 oligomer function. A, in columns 1–5 and 9–13, PA83 or PA63 (1 μg/ml) with or without LF (1 μg/ml) were incubated with VHH (4:1 VHH:toxin) or with medium (columns 1 and 9) for 1 h before the addition to cells. If LF was not present initially, it was present on cells (1 μg/ml) when the antibody-bound PA83 of PA63 was added. Cells were washed after 1 h of toxin binding and incubated overnight in complete medium before viability testing. In parallel plates, the PA83 or PA63 (1 μg/ml) was first bound to cells on ice for 1 h and washed with cold medium before the addition of the first VHH, transfer to 37 °C, and then LF in same amounts described above. Cells stayed at 37 °C for overnight incubation followed by viability assessment. The viability was calculated relative to untreated controls. Data presented in this graph are a representative experiment in which each column is the average of three replicate wells. B, purified PA63 or PA83 were boiled in SDS-loading buffer before SDS-PAGE and Western blotting in a triple sandwich using JKH-C7 as primary antibody, the goat anti-E-tag antibody as a secondary antibody and an IRDye-conjugated anti-goat antibody to detect the E-tag antibody. Western blots are representative of at least two separate experiments.