Gm csf 315 03

Recombinant murine CXCL10 (250-16), CXCL13 (250-24), G-CSF (250-05), and GM-CSF (315-03) were purchased from PeproTech (Rocky Hill, NJ, USA). Directly conjugated anti-mouse mAbs CD11b-FITC (M1/70), CD45-PE/Cy7 (30-F11), Ly6G-APC/Cy7 (1A8), Ly6C-PE (AL-21) were from BD Pharmingen (San Jose, CA, USA). Carboxyfluorescein succinimidyl ester (CFSE) was from Molecular Probes (Eugene, OR, USA). The Annexin-V-APC apoptosis analysis kit (AO2001-11P-G) was obtained from Sungene (Tianjin, China). The mouse cytokine array panel A (ARY006) was purchased from R&D Systems (Minneapolis, MN, USA). The MDSC isolation kit was from Miltenyi Biotec (Bergisch Gladbach, Germany) and the streptavidin particles were from BD IMag (San Jose, CA, USA). Anti-p-Akt (Ser473, #4060), anti-Akt (#4691), anti-p-mTOR (Ser2448, #5536), anti-mTOR (#2983), anti-p-STAT3 (Tyr705, #9145) Abs were purchased from Cell Signal Technology (Danvers, MA). Anti-STAT3 (F-2) Abs was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rapamycin (HY-10219) and Stattic (HY-13818) were purchased from Med Chem Express (New Jersey, USA).

HEK293T cells, Hela cells, iBMDM cells, primary bone-marrow-derived macrophage (BMDM) cells, and peritoneal macrophage (PM) cells were cultured in DMEM medium supplemented with 10% FBS (Gibco, Rockville, MD, USA) and 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C under 5% CO₂ atmosphere. HEK293T cells, Hela cells, and iBMDM cells were maintained in our laboratory. For the isolation of mouse primary peritoneal macrophages (PMs), cells were collected from the lavage of the peritoneal cavity from mice that were pre-stimulated with thioglycollate (TG) for 3 days. For BMDMs, bone marrow cells were isolated from femurs and tibiae of 8-to-12-week-old Otud6a−/− mice and WT mice. Cells were cultured with 20 ng/mL recombinant murine GM-CSF (315-03; Peprotech) in a 10 cm dish for 7 days before experiments.