The turnover of 6myc-HAS2 or K190R mutant 6myc-HAS2, in the absence or presence of USP17/USP17L22, the catalytically inactive C89S mutant USP17 or USP4, was determined by immunoblotting. Protein synthesis was inhibited by treatment with 20 μM cycloheximide (Sigma), followed by incubation for the indicated time periods. The relative 6myc-HAS2 band intensities at 0 h (N0) and later time points (Nt) were quantified, and a half-life was calculated using the formula Nt=N0(1/2)t/t1/2 (www.calculator.net/half-life-calculator ).
The stability of HAS2 was also studied by a pulse-chase assay using metabolic labeling with 35S-methionine/cysteine. HEK293T cells in 6-cm dishes were transfected with the indicated plasmids using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). After 24 h, cells were washed in PBS and starved for 30 min in methionine/cysteine (Met/Cys)-free culture medium (DMEM, Gibco), supplemented with 10 mM HEPES (Sigma). Cells were then incubated for 30 min with 150 μCi [35S]Met/Cys mix (Easy-tag protein labeling mix; Perkin Elmer, Waltham, MA, USA) and chased in DMEM medium supplemented with 10% fetal bovine serum, 2 mM methionine and 2 mM cysteine, for the indicated time periods. Cells were harvested in PBS, snap-frozen in liquid nitrogen and kept at −80 °C until lysis; the amount of HAS2 was determined by immunoprecipitation, followed SDS–PAGE and autoradiography.
The stability of HAS2 was also studied by a pulse-chase assay using metabolic labeling with 35S-methionine/cysteine. HEK293T cells in 6-cm dishes were transfected with the indicated plasmids using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). After 24 h, cells were washed in PBS and starved for 30 min in methionine/cysteine (Met/Cys)-free culture medium (DMEM, Gibco), supplemented with 10 m