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. 2019 Dec 16;117(1):584–594. doi: 10.1073/pnas.1909879116

Fig. 2.

Fig. 2.

FBXW7 regulates STAT2 protein stability. (A and B) WB analysis of WCLs in HeLa (A), WM2664, and A375-SM (B) cells treated with the indicated dose of MLN4924 or MG132 for 6 h. Graphs: Band intensities of STAT2 by 3 independent experiments; values: ±SEM; significance: *P < 0.01 versus nontreated control by Student’s t test. (C) WB analysis of WCLs derived from U2OS cells transfected with Myc-FBXW7. The cells were treated with CHX for 3 h before harvesting. (D) WB analysis of WCLs derived from HeLa cells infected with sh-RNA lentiviral vectors against for cullin 1. After infection, the cells were treated with 2 μg/mL puromycin for 3 d to eliminate noninfected cells. Graph: Band intensities of STAT2 by 3 independent experiments; values: ±SEM; significance: *P < 0.01 versus empty vector (EV) by Student’s t test. (E) WB analysis of WCLs derived from FBXW7 WT (HCT116FBXW7+/+) and FBXW7 knockout (HCT116FBXW7−/−) HCT116 cells. (F) Real-time PCR analysis of STAT2 mRNA expression in HCT116FBXW7+/+ and HCT116FBXW7−/− cells. (G) WB analysis of WCLs derived from HCT116FBXW7+/+ and HCT116FBXW7−/− cells. The cells were treated with 10 μg/mL CHX and harvested at the indicated time points. Graph: Normalized band intensities of STAT2 by 3 independent experiments; values: ±SEM; significance: *P < 0.01 versus nontreated control by Student’s t test. (H) WB analysis of WCLs derived from HCT116FBXW7+/+ and HCT116FBXW7−/− cells. The cells were treated with 10 μM MG132 for 8 h before harvesting. (I) WB analysis of WCLs derived from HCT116FBXW7+/+ and HCT116FBXW7−/− cells. The HCT116FBXW7−/− cells were transfected with Flag-FBXW7 plasmids. (J) WB analysis of WCLs and IPs derived from HCT116FBXW7+/+ and HCT116FBXW7−/− cells. The cells were transfected with His-STAT2 and HA-Ubi. After incubation for 24 h, the cells were treated with 10 μM MG132 for 5 h before harvesting. (K) WB analysis of WCLs derived from 293T cells infected with sh-RNA lentiviral vectors against for FBXW7. After infection, the cells were treated with 2 μg/mL puromycin for 3 d to eliminate noninfected cells. (L) WB analysis of WCLs and IPs derived from the FBXW7 knocked down 293T cells transfected with HA-Ubi. After incubation for 24 h, the cells were treated with 10 μM MG132 before harvesting. (M) WB analysis of WCLs and IPs derived from 293T cells transfected with Xp-STAT2, HA-Ubi, and Myc-FBXW7. After incubation for 24 h, the cells were treated with 10 μM MG132 before harvesting. The specific K48-linked or K63-linked ubiquitination was also detected by WB using the specific antibodies.