Figure 3

AZ2 accelerates c-Myc degradation by the proteasome without ubiquitination. (a) 293-F cells were cotransfected with HA-c-Myc and either HA vector, HA-AZ1, or HA-AZ2. After 24 h, cells were treated with 30 μg/ml cycloheximide and incubated for the indicated times. c-Myc and AZ levels in whole cell extracts were detected by immunoblotting using anti-HA antibody (upper panels). β-Actin bands are also shown. The percentage of c-Myc remaining was quantitated by image analysis and best-fit exponential lines are shown (bottom graph). (b) Putrescine (Put, final 2 mM) was added to the medium 60 min before or simultaneously with cycloheximide. Endogenous c-Myc level in whole cell extracts was detected by immunoblotting using antic-Myc antibody (upper panels). In one series, MG132 (20 µM) was also added 6 h before cycloheximide. (c) 293-F cells were transfected with control siRNA (50 nM) or AZ1 or AZ2 siRNA (20 nM). After 48 h, putrescine (2 mM) was added and after 60 min, c-Myc degradation assays were performed and shown as in (b). (d) Panc-1 cells were transfected with control siRNA (50 nM) or AZ1, AZ2, or Fbxw7 siRNA (20 nM). After 48 h, c-Myc degradation assays were performed and shown as in (b). (e) 293-F cells were transfected with plasmids with mouse cDNA of HA-c-Myc or HA-c-Myc (T58A/S62A) in combination either HA-AZ2 or HA-FBW7. Degradation assay of c-Myc and c-Myc (T58A/S62A) was performed and shown as in (a). (f) Untransfected 293-F cells or 293-F cells transfected with HA-AZ2 for 18 h were treated with E1 inhibitor, PYR-41 (40 μM) for 6 h. Endogenous c-Myc was detected as in (b) (upper panels). Data in a-f represent the mean ± SD calculated from three independent experiments.