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

Fig. 6.

Fig. 6.

UVB promotes destabilization of STAT2 by inducing the STAT2 and FBXW7 interaction. (A) Five-year survival probability of melanoma patients depending on STAT2 protein expression. (B) WB analysis of WCLs derived from HaCaT cells treated with UVB (100 J/m2). The cells were harvested at the indicated time point. (C) WB analysis of WCLs and IPs derived from 293T cells transfected with Xp-STAT2, Myc-FBXW7. The cells were treated with UVB (100 J/m2) and cultured for 6 h before harvesting. (D, Left) WB analysis of WCLs and endogenous STAT2 and FBXW7 IPs derived from HaCaT cells treated with UVB (100 J/m2) and cultured for 4 h before harvesting. (Right) WB analysis of WCLs and endogenous IPs to determine the interaction of STAT2 and FBXW7 by UVB stimulation in HS294T and WM2664 melanoma cells. (E) WB analysis of WCLs derived from HCT116FBXW7+/+ and HCT116FBXW7−/− cells treated with UVB (100 J/m2). The cells were harvested at the indicated time point after UVB treatment. Graph: Normalized band intensities of STAT2 by 3 independent experiments; values: ±SEM; significance: *P < 0.01 versus nontreated control by Student’s t test. (F) WB analysis of WCLs derived from HCT116FBXW7+/+ and HCT116FBXW7−/− cells treated with UVB (100 J/m2) and cultured for 6 h in the presence of 10 μM MG132 before harvesting. (G) WB analysis of WCLs and IPs derived from 293T cells transfected with HA-Ubi, and Myc-FBXW7 together with Xp-STAT2-WT, or Xp-STAT2-4A. The cells were treated with UVB (100 J/m2) and cultured for 6 h before harvesting. (H) WB analysis of WCLs derived from 293T cells transfected with His-STAT2-WT, His-STAT2-4A, and Myc-FBXW7. The cells were treated with UVB (100 J/m2) and harvested at the indicated time point. Graphs: Normalized band intensities of STAT2 by 3 independent experiments; values: ±SEM; significance: *P < 0.01 versus nontreated control by Student’s t test. (I) WB analysis of WCLs derived from 293T cells treated with UVB (100 J/m2) and/or 10 μM MG132. The cells were harvested at the indicated time point. Graph: Normalized band intensities of STAT2 by 3 independent experiments; values: ±SEM; significance: *P < 0.01 versus nontreated control by Student’s t test. (J) Effect of GSK3 inhibitor on the restoration of STAT2 protein levels decreased by UVB stimulation by Western blotting. The cells were harvested at the indicated time point after UVB stimulation either presence of CHIR99021 or absence. (K) WB analysis of cytosol and nuclear fraction to determine the subcellular organelle that mainly host the restoration of STAT2 by MG132 in HS294T melanoma cells.