Fig. 2

ATF2 is a bona fide substrate of the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. a Western blot of WCL from 293 T cells transfected with the indicated plasmids. and treated with MG132 (20 μM), Bortezomib (200 nM), Chloroquine (100 mM) or DMSO for 8 h. Actin was used as a loading control. b Western blot of WCL of 293 T cells transfected with indicated plasmids. c Western blot of WCL of C4–2 cells transfected with indicated plasmids. d Western blot of WCL of 293 T cells transfected with indicated plasmids. e Western blot of the WCL of C4–2 infected with control or lentivirus expressing SPOP-specific shRNAs (shSPOP#1,2). f Quantitative RT-PCR measurement of SPOP and ATF2 mRNA levels in SPOP-depleted C4–2 cells. GAPDH mRNA levels were used for normalization. Standard deviation (S.D.) of at least three independent experiments is shown to indicate statistical significance. **p<0.01. g Western blot of WCL of C4–2 cells infected with control or lentivirus expressing SPOP-specific shRNAs and then treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points (upper panel). At each time point, the intensity of each BET protein was normalized to the intensity of actin and then to the value at 0 h (lower panel). h Western blot of the WCL of C4–2 cells infected with control or lentivirus expressing CUL3 or RBX1-specific shRNAs (sh-CUL3#1,2; sh-RBX1#1,2). i Western blots of the products of in vivo ubiquitination assays performed using cell lysate from 293 T cells transfected with the indicated plasmids and treated with 20 μM MG132 for 8 h