FIGURE 4.

Synergistic inhibitory effects of inhibitors to p97/VCP and importin β on dislocation and ubiquitination of NHK. A, HeLa cells expressing SP-S11-NHK-HA were treated with importazole (IPZ) or DBeQ for 4 h as indicated. Treated cells were then processed for IB. B, HeLa cells were treated with MG132 (10 μm) or different amounts of IPZ or DBeQ as indicated for 4 h before processed for IB. C, HeLa cells stably expressing SP-S11-NHK-HA and S1-10 were treated with MG132 (10 μm) alone or MG132 (10 μm) together with IPZ (30 μm) and/or DBeQ (7.5 μm) for 4 h. The drGFP images were taken under the same parameters. D, HeLa cells stably expressing SP-S11-NHK-HA and S1-10 were treated as in C. The relative fluorescence units (RFU) of drGFP were measured with a fluorescence microplate reader. The data are presented as the means ± S.E. of 4 samples. The drGFP intensity increased after 4-h treatment with MG132 was set as 1. E, non-transfected HeLa cells or HeLa cells expressing SP-S11-NHK-HA were treated with inhibitors as indicated for 4 h. The IP and IB were performed as in Fig. 3. The numbers in PolyUb blot (lanes 9–12) indicate the relative density of polyubiquitinated S11-NHK-HA. F, as in E, except that HeLa cells expressing S11-NHK-HA (no SP) were used. WCL: whole cell lysate.