Puromycin p8230

OVCAR-3 cells were cultured in RPMI-1640 (Gibco, NY, USA) containing 10% fetal bovine serum (FBS; Cellmax, Lanzhou, China) and 1% antibiotic mixture (Beyotime, Shanghai, China) at 37 °C in a humidified incubator with 5% CO₂. A total of 3 × 10⁵ cells were infected with a lentivirus (Sunbio Medical Biotechnology, Shanghai, China) carrying the SPOP gene (ov-SPOP), SPOP shRNA (sh-SPOP), or negative control (ov-NC or sh-NC) following the manufacturer’s instructions. The infected cells were selected with 72 h of incubation with 10-µg/mL puromycin (P8230; Solarbio, Beijing, China).

Antibodies against HA-Tag Rabbit (C29F4, #3724), HA-Tag mouse (6E2, #2367), acetyl-lysine (#9441), p-P38 (Thr180/Tyr182) (D3F9, #4511), P38(#9212), p-ERK (E-4, #sc-7383), ERK (#4696), p-AKT (Ser473, 193H12, #4058), AKT (#9272), p-mTOR (Ser2448, #2971), mTOR (#2983), p-p70 S6K (Thr421/Ser424, #9204), S6K (#9202), and UBC9 (D26F2, #4786) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against SENP1 (A1260) were purchased from ABclonal (Woburn, MA, USA). Antibodies against Flag-tag (F1804), MG132, and cycloheximide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against Sirt2 (#ab67299) and SUMO1 (#Y299) were purchased from Abcam (Cambridge, UK). Puromycin (P8230) and rapamycin (R8140) were purchased from Solarbio (Beijing, China). Regents of SB202190 (#S1077), LY294002 (#S1105), NAD⁺ (#S2518), trichostatin A (TSA) (#S1045), nicotinamide (NAM) (#S1899), and protease inhibitor cocktail (EDTA-Free,100 × in DMSO) were obtained from Selleck (Houston, TX, USA). The KOD-plus-mutagenesis kit was purchased from Toyobo (Osaka, Japan). Protein A/G magnetic beads were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ni-NTA beads were purchased from Qiagen (Hilden, Germany).