Figure 5. Extracellular Hsp90 serves as a co-factor for KSHV-induced MAPK activation.

(A) HeLa cells were pre-treated with the indicated concentrations of DNo for 16 h. Immunoblot analyses were performed 16 h later for total (t) or phosphorylated (p) signaling intermediates or β-Actin as an internal control. Control cells were treated with 10 μM of the MAPK-specific inhibitor U0126 for 1.5 h at 37°C. (B) Hela cells and pDMVEC were incubated with 1.0 μM DNo for 16 h beginning immediately following KSHV incubation, then immunoblot analyses were performed for signaling molecules as above. (C) HeLa cells were incubated with 30 μg/ml of either control isotype Ab (isoAb) or monoclonal anti-Hsp90 antibodies (771Ab and 840Ab) for 12 h at 37°C, then with purified KSHV for 2 h. Immunoblot analyses were performed 16 h later for signaling molecules as above. (D) HeLa cells were incubated with KSHV with or without pre-incubation with 30 μg/ml anti-Hsp90 antibodies (840Ab). Immunoblot analyses were performed 16 h later. (E) HeLa cells were incubated with DMSO or 0.5 μM DNo for 16 h, or first transiently transfected with 1 μg of pcDNA3.1 control (pc), pcDNA3.1-FLAG-MEK (pcMEK), pcDNA3.1-FLAG-ERK (pcERK) or pcDNA3.1-FLAG- dominant-negative-ERK (pcERK-DN) vectors prior to DNo treatment. 24 h post-transfection, cell lysates were collected and analyzed as above.