Anti usp7 antibody

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics. iSLK cells were cultured with the same medium plus 450 µg/mL G418 and 1 µg/mL puromycin [34 (link)]. The generations of iSLK.BAC16, iSLK.BAC16-ORF45 ΔC19, and iSLK.BAC16-ORF45 ΔC19R cells were described previously [20 (link),35 (link),36 (link)]. These cells were cultured similarly to iSLK with the addition of 400 µg/mL hygromycin B. Anti-HA, anti-FLAG M2, anti-GST antibodies, and EZview Red anti-FLAG M2 resin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-USP7 antibody was purchased from Bethyl Laboratories (Montgomery, TX, USA). Antibodies that detect ORF45 were generated as previously described [11 (link),37 (link)]. Monoclonal antibodies against ORF26, ORF33, and ORF38 were generated as described [21 (link)]. Protein-G beads were purchased from Thermo Fisher Scientific (Waltham, MA, USA). EZ-Run Pre-Stained Protein Ladder was purchased from Fisher Scientific (Pittsburgh, PA, USA). TAT-C19 and TAT were synthesized by Biomatik (Wilmington, DE, USA).

Total proteins were extracted in 50 mM Tris–HCI pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1% Triton X100 and 2× Protease inhibitors (Pierce #78442) and briefly sonicated. The endogenous USP7 was immunoprecipitated by overnight incubation at 4°C of 2 mg protein lysate with 6 μg anti-USP7 antibody (Bethyl A300-033A) followed by 1h30 incubation with Protein A/G magnetic beads (Pierce #88802) at 4°C. Protein complexes were washed 5 times with 50 mM Tris–HCI pH 7.5, 250 mM NaCl, 0.5 mM EDTA, 1% Triton X100, 2× protease inhibitors (Pierce #78442) and eluted in 2× SDS gel-loading buffer for 10 min at 95°C. Usp7-KO ES cells were used as negative controls for the immunoprecipitation experiments. For Western blot analysis, 15% of eluted proteins and 30 μg of input proteins were loaded on SDS-polyacrylamide gels.