Fig. 2

SPOP promotes Caprin1 degradation and ubiquitination. a Western blots of indicated proteins in WCLs from C4–2 cells treated with DMSO, MG132 (20 μM) or with MLN4924 (100 nM) for 8 h. b RT-qPCR assessment of Caprin1 mRNA expression in C4–2 cells treated with DMSO, MG132 (20 μM) or with MLN4924 (100 nM) for 8 h. The mRNA level of GAPDH was used for normalization. Data are shown as means ± SD (n = 3). c Western blot of indicated proteins in WCLs from C4–2 cells transfected with indicated siRNAs. d Western blot of indicated proteins in WCLs from 293 T cells transfected with indicated plasmids with DMSO, MG132 (20 μM) or with Bortezmib (20 nM) for 8 h. e Western blot of indicated proteins in WCLs from 293 T cells transfected with indicated plasmids. f Western blot of indicated proteins in WCLs from FLAG-SPOP inducible Flp-In T-Rex 293 cells treated with tetracycline with indicated times. g Western blot of indicated proteins in WCLs from C4–2 cells with SPOP knockout through CRISPR/Cas9 methods. Parental C4–2 cells were used as the control; Western blot of indicated proteins in WCLs from C4–2, LNCaP and 22Rv1 cells infected with lentivirus expressing SPOP-specific shRNA or negative control (NC). h RT-qPCR measurement of Caprin1 mRNA expression in C4–2 cells infected with lentivirus expressing SPOP-specific shRNA or NC; RT-qPCR measurement of Caprin1 mRNA expression in parental and SPOP-KO C4–2 cells. Data are shown as means ± SD (n = 3). i, j Western blot of indicated proteins in WCLs of C4–2 cells infected with lentivirus expressing SPOP-specific shRNA or NC for 48 h and then treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points (i). At each time point, the intensity of Caprin1 was normalized to the intensity of actin and then to the value at 0 h (j). Similar results were obtained from two independent experiments. k, l Western blot of the products of in vivo ubiquitination assays from 293 T cells transfected with the indicated plasmids and treated with 20 μM MG132 for 8 h