Figure 1
From: RUNX family members are covalently modified and regulated by PIAS1-mediated sumoylation
Mammalian PIAS1 sumoylates RUNX family members. (a) HA-tagged human PIAS1, PIAS2α, PIAS12β, PIAS3 or PIAS4 were coexpressed with Myc-tagged RUNX3 in HEK293 cells. The RUNX3-PIAS interaction was measured by immunoprecipitation35 and IB. (b) HA-PIAS1 or HA-PIAS1-C351A (defective in SUMO E3 ligase activity) was coexpressed with FLAG-SUMO1 and Myc-RUNX3, and RUNX3 sumoylation and the RUNX3-PIAS1 interaction were analyzed by IP and IB. (c) HA-PIAS1 and FLAG-SUMO1 were expressed in HEK293 cells. Sumoylation of endogenous RUNX3 was detected by IP with anti-RUNX3 antibody (5G4) and IB with anti-FLAG antibody. (d) HEK293 cell were transfected with FLAG-SUMO1 and treated with si-PIAS1 (100 nM). Two independent si-PIAS1 RNAs #1 and #2 were used. Sumoylation of endogenous RUNX3 was detected by IP with anti-RUNX3 antibody (5G4) and IB with anti-FLAG antibody. (e) Myc-tagged RUNX1, RUNX2 or RUNX3 were coexpressed with HA-PIAS1 and FLAG-SUMO1 (wild-type (G) or G>A mutant (A)), and sumoylation of the RUNX family members was analyzed by IB. (f) FLAG-tagged SUMO1, SUMO2 and SUMO3 were coexpressed with HA-PIAS1 and Myc-RUNX3. Lysates were analyzed by IB using the indicated antibodies.
