Park et al. 10.1073/pnas.0609852104.

Supporting Information

Files in this Data Supplement:

SI Figure 5
SI Figure 6
SI Figure 7
SI Figure 8




SI Figure 5

Fig. 5. IRF-1 is SUMOylated in MCF7 cells. Immunoprecipitation with an antibody specific for SUMO-1, followed by immunoblotting with anti-IRF-1, confirmed that the shifted bands are SUMOylated IRF-1.





SI Figure 6

Fig. 6. In vitro SUMOylation assay with IRF-1 splicing variants. (A) Diagram depicting IRF-1 splicing variants. (B) SUMOylation sites were located within C-terminal domain. The stars indicate the SUMOylated bands.





SI Figure 7

Fig. 7. SUMOylation of IRF-1 antagonizes ubiquitination of IRF-1. HEK293 cells were transfected with Xpress-tagged IRF-1 expression plasmid with or without plasmid encoding SUMO-1 and treated with 10 mM MG132 for 12 h. The proteins were immunopurified with anti-Xpress antibody and then probed with the indicated anti-ubiquitin and anti-SUMO-1 antibodies. The coexpression of SUMO-1 decreased the level of ubiquitinated IRF-1, indicating that SUMOylation of IRF-1 antagonizes its ubiquitination.





SI Figure 8

Fig. 8. SUMO-IRF-1 inhibits the p21 promoter induced by IRF-1. (A) The SUMOylation-deficient IRF-1 mutant confers increased transactivation activity. HEK293T cells were transfected with 200 ng of p21-Luc and the increasing concentrations of plasmid encoding IRF-1 or IRF-1 mutant (K275,299R). Cell lysates were probed with anti-IRF-1 antibodies to verify the level of IRF-1 proteins. (B) SUMO-IRF-1 fails to inhibit the p21 promoter activity induced by p53. HEK293 cells were cotransfected with 300 ng of p21-Luc reporter and 300 ng of an IRF-1 expression plasmid (pcDNA3/IRF-1) in the presence or absence of 300 ng SUMO-IRF-1.