Figure 4

Myrf K276 sumoylation represses the transactivation function of NTM. (A) The transcriptional activity of wild-type Myrf was compared with that of mutant ones by luciferase assay in Oli-neu cells. The luciferase activity of Myrf was set to 1, and the reported values are means and standard errors. *p < 0.05 by two-tailed unpaired Student’s t test with Bonferroni correction. The luciferase assay samples were subsequently analysed by Western blot to check protein expression levels. The full blots are shown in Supplementary Figure 4A. (B) Immunoprecipitation experiments showed that K276 undergoes sumoylation in the Gal4 fusion context. The full blot is shown in Supplementary Figure 4B. (C) Luciferase assay showed that K276 sumoylation represses the transactivation function of NTM in the context of Gal4 fusion proteins. NTM-K276R-G4 is the same as NTM-G4 (Fig. 2A) except for the K276R mutation that prevents K276 sumoylation. Shown are means and standard errors. *p < 0.001 by two-tailed unpaired Student’s t test with Bonferroni correction. The luciferase assay samples were subsequently analysed by Western blot to check protein expression levels. The grouping of blots cropped from different parts of the same gel is indicated by dividing lines. The full blots are shown in Supplementary Figure 4C. (D) The K276R mutation does not affect the stability of Myrf N-terminal fragment. Oli-neu cells were transfected with Flag-Myrf or Flag-Myrf-K276R. Transfected cells were aliquoted into small dishes and subject to cycloheximide treatment (25 ug/ml) for different durations to determine the degradation rate of Myrf N-terminal fragment. The grouping of blots cropped from different parts of the same gel is indicated by the dividing line. As indicated in the full blot (Supplementary Figure 4D), the results for Myrf and Myrf-K276R are from different exposure times (2 min and less than 1 min, respectively). IB: immunoblotting. IP: immunoprecipitation.