Figure 3.

Effect of Hsp90 inhibitors on ER-to-Golgi transport of VSV-G^ts. (A) GA does not inhibit NSF or p97 ATPase activity. ATPase activity of NSF or p97 was measured in the absence of presence of GA (50 μM) (see Materials and Methods). Quantitation is based on two independent experiments with triplicate samples. Error bars indicate SEM. (B) VSV-G^ts transport was measured using a temperature-shift, pulse-chase protocol as described in Materials and Methods. After transport, VSV-G^ts was immunoprecipitated, samples were digested with endo H and processed for SDS-PAGE. (Top) Shown are autoradiographs of processing intermediates of VSV-G^ts formed in the presence of different inhibitors following 30 min of transport at the indicated final concentrations of the indicated inhibitor. After endo H digestion, VSV-G^ts migrates on SDS-PAGE as the endo H-sensitive ER glycoform (S); an intermediate early (cis/medial-) Golgi glycoform (I), or a late (trans-) Golgi endo H-resistant mature glycoform (R). (Bottom) The ratio of the R form to total amount of VSV-G^ts detected in all glycoforms was quantified and normalized with respect to the vehicle (DMSO) control to show the relative inhibitory effect of Hsp90 inhibitors on ER-to-Golgi transport.