K63 specific polyubiquitin

Murine bone marrow-derived macrophages (BMMs) were isolated, as described previously [24 (link)]. In brief, bone marrow cells were flushed from the femora and tibiae of 8-week-old C57BL/6 mice and cultured for 2 days with complete α-MEM medium containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 30 ng/mL M-CSF. Cells in the supernatant were collected and cultured in the above medium for 2 days. Adherent cells were considered to be BMMs. GH3 and 293T cells were cultured in completed DMEM supplemented with 10% fetal bovine serum. A miR-19a mimic and a scrambled control were synthesized by Tsingke Biotechnology Company (Beijing, China). Lentivirus expressing CYLD and scrambled control virus were purchased from Vigene Biosciences (Rockville, MD, USA) and transfected by Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). Recombinant mouse M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN, USA). Primary antibodies included anti-GAPDH, CYLD (ProteinTech Group, Chicago, IL, USA), TRAF6, TRAP, K63-specific polyubiquitin, NFATc1, c-Fos, AP-1, p65, p-p65, NF-кB, p38, p-p38, JNK, p-JNK, ERK, and p-ERK (Cell Signaling Technology, Beverly, MA, USA).

All ligands, LPS, Pam3CSK4 (PAM), and R848, were purchased from InvivoGen (San Diego, CA, USA). Ubiquitin ligase inhibitor (ULI) small-molecule enhancer of rapamycin (SMER) 3 was purchased from Selleckchem (Houston, TX, USA). Protein A-Sepharose beads were purchased from Sigma (St. Louis, MO, USA). Phospho-specific antibodies against ERK1/2, p38, c-Jun NH(2)-terminal kinase (JNK), TAK1, as well as TRAF6, K63-specific polyubiquitin and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p38 and polyubiquitin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG secondary antibodies were purchased from Cell Signaling Technology, and HRP-conjugated anti-goat antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and FITC-labeled anti-CD11b was purchased from BD Biosciences (San Jose, CA, USA).