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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: Cell Microbiol. 2019 Jul 5;21(10):e13074. doi: 10.1111/cmi.13074

FIGURE 5.

FIGURE 5

Hsp90 is required for PtxSl delivery to the cytosol. (a) Hsp90 was perfused over a PtxSl-coated SPR sensor at 37°C in the absence or presence of ATP. (b) CHO cells pulse-loaded with 1 μg/mL of Ptx at 4°C were chased at 37°C for 3 h in toxin-free medium. The cytosolic extracts from digitonin-permeabilized cells were then perfused over an SPR sensor appended with an anti-PtxSl antibody. Cells were untreated (no treatment), treated with the Hsp90 inhibitor geldanamycin (GA), or treated with a drug that blocks toxin transport to the ER (brefeldin A; BfA). A cell extract from unintoxicated CHO cells was also perfused over the sensor, as were 1 ng/mL and 10 ng/mL PtxSl standards. (c) CHO cells mock transfected or transfected with an ssPtxSl expression plasmid were chased for 24 h in the absence or presence of the Hsp90 inhibitor GA. Separate organelle (pellet; P) and cytosolic (supernatant; S) fractions generated from digitonin-permeabilized cells were then probed by Western blot with a rabbit anti-PtxS1 primary antibody and HRP-conjugated goat anti-rabbit IgG secondary antibody. One of two representative experiments is shown.