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).
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