FIG 1.

K114 and K137 of SOCS1 are target sites for K48-linked ubiquitination. (A) Full-length sequence of SOCS1. Lysine residues are in bold red. (B) HEK 293T cells were transfected with HA-Ub-K48 (1.5 mg/well) together with WT or SOCS1 point mutant plasmids (1.5 mg/well). Thirty-six hours later, the cell lysates were immunoprecipitated (IP) with anti-Myc, and ubiquitinated SOCS1 was detected by immunoblotting (IB) with anti-HA. WCL, whole-cell lysate. (C and D) HEK 293T cells or DEFs were transfected with the WT or the K114R or K137R mutant (2.5 mg/well). Thirty-six hours later, the cells were treated with CHX (200 μg/mL). Cells were harvested 0 h, 4 h, 8 h, and 12 h after treatment. The protein level of WT or mutant SOCS1 was determined by IB with anti-Myc. (E) HEK 293T cells were transfected with HA-Ub-K48 (1.5 mg/well) together with the WT, the K114R or K137R single-point mutant, or the DM (1.5 mg/well). Thirty-six hours after transfection, the cell lysates were immunoprecipitated with anti-Myc, and ubiquitinated SOCS1 was detected by IB with anti-HA. (F) HEK 293T cells were transfected with the WT or DM (2.5 mg/well). Thirty-six hours later, the cells were treated with CHX (200 μg/mL). Cells were harvested 0 h, 4 h, 8 h, and 12 h after treatment. The protein level of WT or mutant SOCS1 was determined by IB with anti-Myc. The densitometric values of the SOCS1 protein were quantified and analyzed. All data are the mean values obtained from at least three replicates of independent experiments.