Figure 4
From: Structure-function analysis indicates that sumoylation modulates DNA-binding activity of STAT1
Sumoylation does not prevent dimerization of STAT1. (A) Sumoylated STAT1 can participate in STAT1 dimers. Cos-7 cells were transiently transfected with STAT1-Flag, STAT1-HA and SUMO-1-His as indicated. 48 hours after transfection cells were either treated or left untreated with 100 ng/ml of human IFN-γ for 45 min and with 0.3 M sorbitol for osmotic shock for 15 min followed by cell lysis. Equal amounts of cell lysates were subjected to co-immunoprecipitation with anti-Flag agarose beads followed immunodetection with anti-HA antibody to reveal STAT1-Flag interacting STAT1-HA proteins. Equal amounts of whole cell lysates (1% input) were separated on the same SDS-PAGE gel and immunoblotted with anti-HA (right panel). Longer exposure time was needed to reveal STAT1-sumoylation bands in the inputs. (B) Control experiment showing that anti-Flag agarose does not bind directly to HA-tagged STAT1. Cos-7 cells were transfected, stimulated and immunoprecipitated as described above. IB: immunoblot, IP: immunoprecipitation, OS: osmotic shock.