American Society of Hematology

Figure 1.

$Figure 1. Stat1 SUMO-ylation in HEK-293T cells. (A) HEK-293T cells were transfected with HA–SUMO-1, Ubc9, and/or PIASy. Later (48 hours), they were stimulated with IFN-γ (3 ng/mL for 30 minutes). WCEs were immunoprecipitated with an anti-Stat1 antibody and sequentially immunoblotted with HA and Stat1 antibodies. (B) Left panel: 48 hours after transfection with HA–SUMO-1 and Ubc9, HEK-293T cells were stimulated with IFN-γ (3 ng/mL for 30 minutes). WCEs were either precipitated with a biotinylated GAS oligonucleotide (Oligo; Table S1) or Stat1-specific antibody (α-Stat1). Precipitates were fractionated by SDS-PAGE, and sequentially immunoblotted with Stat1-phosphotyrosine (top) and Stat1-specific antibodies (bottom). Right panel: WCEs were prepared 48 hours after transfection with His–SUMO-1 and Ubc9; HEK-293T cells were stimulated with IFN-γ (3 ng/mL for 30 minutes) and either precipitated with a Stat1-specific antibody (anti-Stat1) or ProBond nickel beads, and sequentially immunoblotted with Stat1-phosphotyrosine (top) and Stat1-specific antibodies (bottom). Nonspecific (NS) band is indicated. Data are representative of 3 independent studies.$

Stat1 SUMO-ylation in HEK-293T cells. (A) HEK-293T cells were transfected with HA–SUMO-1, Ubc9, and/or PIASy. Later (48 hours), they were stimulated with IFN-γ (3 ng/mL for 30 minutes). WCEs were immunoprecipitated with an anti-Stat1 antibody and sequentially immunoblotted with HA and Stat1 antibodies. (B) Left panel: 48 hours after transfection with HA–SUMO-1 and Ubc9, HEK-293T cells were stimulated with IFN-γ (3 ng/mL for 30 minutes). WCEs were either precipitated with a biotinylated GAS oligonucleotide (Oligo; Table S1) or Stat1-specific antibody (α-Stat1). Precipitates were fractionated by SDS-PAGE, and sequentially immunoblotted with Stat1-phosphotyrosine (top) and Stat1-specific antibodies (bottom). Right panel: WCEs were prepared 48 hours after transfection with His–SUMO-1 and Ubc9; HEK-293T cells were stimulated with IFN-γ (3 ng/mL for 30 minutes) and either precipitated with a Stat1-specific antibody (anti-Stat1) or ProBond nickel beads, and sequentially immunoblotted with Stat1-phosphotyrosine (top) and Stat1-specific antibodies (bottom). Nonspecific (NS) band is indicated. Data are representative of 3 independent studies.

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