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

Fig. 1.

Fig. 1.

STAT2 is a binding partner of FBXW7. (A) Screening of FBXW7 binding partner via a mammalian 2-hybrid assay in 293T cells transfected with the indicated plasmids. The binding affinity was analyzed via a luciferase activity. Relative luciferase activity (fold-change) was normalized against control (pBIND-Gal4-FBXW7+pACT-VP16-mock). Data: Triplicate experiment; values: ±SEM; significance: *P < 0.01 versus control by Student’s t test. (B) Western blot (WB) analysis of whole-cell lysates (WCLs) and anti-Xp IPs derived from 293T cells transfected with Xp-STAT2 and Myc-FBXW7 plasmids. After 24 h of incubation, the cells were treated with 10 μM MG132 for 4 h before harvesting. (C) WB analysis of WCLs and co-IPs of endogenous STAT2 and FBXW7 was conducted in HeLa cells using anti-STAT2 antibody. (D) Subcellular localization of STAT2 and FBXW7 proteins. (Left) Western blotting using cytosol and nuclear fraction. (Right) Immunocytofluorescence assay using specific antibodies as indicated (400×). (E) WB analysis of WCLs and anti-Myc IPs derived from 293T cells transfected with His-STAT2 and Myc-cullins (cullin 1, 2, 3, 4A, 4B, and 7) plasmids. After 24-h incubation, the cells were treated with 10 μM MG132 for 4 h before harvesting. (F) WB analysis of WCLs and anti-Xp IPs derived from 293T cells transfected with Xp-STAT2 and Myc-RBX1 plasmids. After 24 h of incubation, the cells were treated with 10 μM MG132 for 4 h before harvesting. (G) WB analysis of WCLs and anti-Xp IPs derived from 293T cells transfected with Xp-STAT2 and Gal4-tagged F-box proteins. After 24 h of incubation, the cells were treated with 10 μM MG132 for 4 h before harvesting.