Figure 3
From: RUNX family members are covalently modified and regulated by PIAS1-mediated sumoylation
PIAS1-mediated sumoylation inhibits transactivation activity of RUNX3. (a) Myc-RUNX3, FLAG-SUMO1 and HA-PIAS1 were transfected into HEK293 cells as indicated. The reporter activity of 6xOSE promoter luciferase was measured with a luminometer and normalized to that of pCMV-β-galactosidase, which was included as an internal control. *P<0.05 by Student’s t-test. (b) Myc-RUNX3-WT or Myc-RUNX3-K148R were co-transfected with FLAG-SUMO1 and HA-PIAS1 into HEK293 cells as indicated. The reporter activity of 6xOSE promoter luciferase was measured and normalized to that of pCMV-β-galactosidase. *P<0.05. (c) Myc-RUNX3 was co-transfected with FLAG-SUMO1 and HA-PIAS1 into HEK293 cells as indicated. The reporter activity of p21-promoter-luciferase (Chi et al.25) was measured and normalized to that of pCMV-β-galactosidase. *P<0.05. (d) Myc-RUNX3 (WT) or Myc-RUNX3-K148R (KR) was co-transfected with FLAG-SUMO1 and HA-PIAS1 into HEK293 cells as indicated. The levels of the expressed proteins and endogenous p21 were measured by IB. (e) Myc-RUNX3 (red) or Myc-RUNX3-K148R (red) was expressed with or without HA-PIAS1 (green) in HeLa cells, and the subcellular localization of each protein was visualized by immunostaining. Nuclei were stained with DAPI. (f) Myc-RUNX3-WT and Myc-RUNX3-K148R were coexpressed with increasing amounts of FLAG-CBFβ2 in HEK293 cells. The RUNX3-CBFβ2 interaction was analyzed by IP and IB. (g) Fixed amounts of Myc-RUNX3-WT, HA-PIAS1 and FLAG-SUMO1 were coexpressed with increasing amounts of HA-CBFβ2 in HEK293 cells. RUNX3 sumoylation was analyzed by IP and IB.
