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. 2015 Oct 29;5:15768. doi: 10.1038/srep15768

Figure 5. 5AZ potentiates sumoylation of IRF1.

Figure 5

(A) NIH3T3 cells were transfected with flag-SUMO-1 and c-myc-IRF1 for 24 hours and were then treated with LPS with or without 5AZ for a further 24 hours. Cell extracts were blotted with anti-IRF1 antibody. Longer exposure revealed sumoylated IRF1 bands. (B) NIH3T3 cells were transfected with flag-SUMO-1 and c-myc-IRF1 for 24 hours and were then treated with LPS with or without 5AZ for a further 24 hours. Cell extracts were precipitated with anti-SUMO-1 antibody and blotted with anti-IRF1 or anti-flag antibody. (C) Ubc9 protein level was reduced by LPS treatment, while upregulated in the presence of 5AZ. On the other hand, PIAS3 did not show a significant change. (D) HeLa cells were transfected with flag-SUMO-1, c-myc-IRF1, and control siRNA or UBC9 siRNA for 24 hours and were then treated with LPS with or without 5AZ for a further 24 hours. Cell extracts were blotted with anti-IRF1 antibody. Longer exposure revealed sumoylated IRF1 bands. (E) RAW264.7 cells were transfected with siRNA control (siCon) or siRNA Ubc9 (siUbc9) for 24 hours, and further treated with LPS with or without 5AZ. Immunoblots were performed and quantitated. *Significantly different from siCon-transfected cells treated with LPS, p < 0.05. #Significantly different from siCon-transfected cells treated with LPS and 5AZ, p < 0.05. (F) c-myc-IRF1 and flag-SUMO-1 were transfected into 293T cells. Cells were treated with LPS or LPS + 5AZ for 24 hours, and localization of IRF1 or flag was visualized by fluorescence immunostaining.