FIGURE 4.

JKH-C7 does not prevent PA binding or cleavage and inhibits oligomer endocytosis. Various PA83, mutant PA proteins, or PA63 (1 μg/ml) were preincubated with antibodies (VHH:toxin, 4:1) in serum-free DMEM for 30 min to 1 h before the addition to CHO WTP4 cells for 60 min. Cells were either lysed directly in radioimmune precipitation assay buffer without trypsinization or trypsinized as described under “Experimental Procedures” to remove all cell surface proteins before lysis. Western blotting was performed with anti-PA polyclonal (1:5000), and in B–E, the blots were re-probed with goat anti-E-tag antibody (1:1000) to detect the JKH-C7 VHH in or on cells. IR-dye-conjugated secondary antibodies of different wavelengths were used to detect the PA and anti-E-tag antibodies in panels B and D where the re-probed section of the blot is shown as a separate panel. In panels C and E a single wavelength IR-dye secondary was used for detection of both primary antibodies, and the whole re-probed blot is shown. In panel A both a color and black and white image for the same blot probed with a single IR-dye secondary is shown for better identification of oligomer and lanes. CR stands for “cross-reactive” band, which serves as a nonspecific equal loading control. Western blots are representative of at least two separate experiments.