Figure 4

The small molecules did not inhibit ubiquitination of APOBEC3G or general proteasomal activity. A. 293 T cells were transfected with the Vif expression vector, co-transfected with APOBEC3G-myc expression vector or empty vector. 24 hours before harvesting, MM-1, MM-2 or DMSO was added in culture media as indicated. Cells were lysed and immunoprecipitated by anti-myc antibody in the presence of the compound as indicated, and bound protein was analyzed by immunoblotting with anti-Vif and anti-myc sera. B. 293 T cells were transfected with expression vectors for EGFP-fused APOBEC3G and HA-tagged ubiquitin, in the absence or presence of co-transfection of the Vif expression vector, and treated with MM-1, MM-2 or DMSO for 24 hours. Cells were then lysed, and immunoprecipitated with anti-GFP rabbit serum. Bound protein was analyzed by immunoblotting with anti-ubiquitin and anti-GFP antibodies. C. An in vitro proteasome 20S assay kit was used for testing inhibitory potential of the compounds to proteasomal activity. DMSO was used as control for the compounds and epoxomicin was used as control for proteasome inhibitor.