Fig. 6

SPOP suppresses the activation of MAPK/ERK cascade. a Western blot of indicated proteins in WCLs from Tet-on-inducible Ishikawa cells treated with DOX. b Western blot of the indicated proteins in WCLs from parental and SPOP-KO Ishikawa cells. c, d EV and SPOP-ΔNLS overexpressed Ishikawa cells were serum-starved for 48 h and then treated with EGF (100 nM) for the corresponding times. The WCLs were prepared for WB analysis. At each time point, the intensity of p-ERK1/2, p-MEK1/2, p-MSK1 was normalized to the intensity of ERK1/2, MEK1/2, MSK1 respectively and then to the value at 0 min d. e, f Parental and SPOP-KO Ishikawa cells were serum-starved for 48 h and then treated with EGF (100 nM) for the corresponding times. The WCLs were prepared for WB analysis. At each time point, the intensity of p-ERK1/2, p-MEK1/2, p-MSK1 was normalized to the intensity of ERK1/2, MEK1/2, MSK1 respectively and then to the value at 0 min f. g, h SPOP-KO Ishikawa cells stably overexpressing BRAF-WT, or T121I mutant were serum-starved for 48 h and then treated with EGF (100 nM) for the corresponding times. The WCLs were prepared for WB analysis. At each time point, the intensity of p-ERK1/2, p-MEK1/2, p-MSK1 was normalized to the intensity of ERK1/2, MEK1/2, MSK1 respectively and then to the value at 0 min h. i, j The WCLs from SPOP-KO Ishikawa cells stably overexpressing EV, SPOP-WT, SPOP- ΔNLS i and mutants j were prepared and subjected to co-IP with anti-BRAF antibody. The immunoprecipitates were analyzed by WB with the indicated antibodies. k The WCLs from BRAF-KO Ishikawa cells stably overexpressing BRAF-WT or T121I mutant were prepared and subjected to co-IP with anti-BRAF antibody. The immunoprecipitates were analyzed by WB with the indicated antibodies. The band intensity of the indicated proteins was first normalized to loading input l and the value was further normalized to the one in cells. All data shown are mean values ± SD (n = 3). The p-values were calculated using the Two-way ANOVA test in c, e, g. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001