FIG. 1.

Hsp90 inhibition suppresses FHV protein A synthesis. (A) Drosophila S2 cells stably transfected with pS2FA (lanes 1 to 4), pS2LacZ (lanes 5 to 7), or pS2FB (lanes 8 and 9) were induced with Cu²⁺ and labeled with 100 μCi per ml [³⁵S]Met-Cys in the presence of 1 μM geldanamycin (GA), 10 μM lactacystin (Lact), or 100 μg per ml cycloheximide (CX) for 90 min. Radiolabeled proteins were immunoprecipitated with either HA-specific antibodies for FHV protein A (PtnA) and β-galactosidase or FHV protein B2 (PtnB)-specific antibodies, separated by SDS-PAGE, and analyzed by fluorography. (B) Quantitative data for the effect of geldanamycin and proteasome inhibitors (PI) on FHV protein A, protein B2, and β-galactosidase synthesis compared to no-inhibitor controls. Proteasome inhibitor results are composite data from experiments using either lactacystin or MG132. (C) Total lysates (lanes 1 to 4) or carbonate-resistant membrane fractions (lanes 5 and 6) from cells metabolically labeled with [³⁵S]Met-Cys in the presence of the inhibitors listed above the lanes. Lanes 1 to 4 correspond to similarly numbered lanes in A. Samples for lanes 5 and 6 were prepared by differential centrifugation as previously described (25) and represent cellular proteins tightly associated with intracellular membranes (5). The asterisk indicates a cellular protein whose degradation is inhibited by lactacystin. Results are representative of three independent experiments, and for B, results represent the means ± standard errors of the means relative to vehicle-only controls. MW, molecular weight (in thousands).