P iκbα

Total lysates of human OA chondrocytes were prepared using RIPA buffer (Biosesang, Gyeonggi-do, Korea). Primary antibodies against MMP3, MMP13 (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA), COL2A1 (1:1000 dilution, Abcam), ACAN (1:500 dilution, Invitrogen), p38, p-p38, ERK, p-ERK, JNK, p-JNK, p65, p-p65, IκBα, p-IκBα, or β-actin (1:1000 dilution, Cell Signaling Technology) and appropriate HRP-conjugated secondary antibodies (1:2000 dilution, Bethyl) were used. Enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific) was used to detect for specific bands. Chemiluminescent signals were analyzed on a ChemiDoc XRS gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA), and intensity of specific bands was quantified using Image J software (NIH, MD, USA).

Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher. SeV and VSV-GFP were described previously^{26 (link),27 (link)}. The cultivation of human embryonic kidney (HEK) 293 T cells have been described previously^{28 (link)}.

BMMs were seeded into six-well plates at the appropriate density and stimulated with 30 ng/mL M-CSF and 50 ng/mL RANKL plus either MSNs (16 or 64 μg/mL) or MSNs-ISL (16 or 64 μg/mL) for 30 min or 24 h. The cells were washed twice with ice-cold PBS and then lysed using RIPA lysate containing protease and phosphatase inhibitor. Protein content was determined using a BCA protein quantification kit (Beyotime Inc, Shanghai, China) following the manufacturer's protocol. Total proteins were transferred onto a polyvinylidene difluoride membrane, blocked in non-fat milk for 1 h at room temperature, and incubated overnight with the specific primary antibodies at 4 °C. GAPDH served as the protein internal standard and the standard for quantifying protein expression. The specific primary antibodies used were p38, p-p38, extracellular signal-regulated protein kinase (ERK), p-ERK, nuclear factor-κB (NF-κB) p65, p NF-κB p65, inhibitor of κBα (IκBα), p-IκBα, GAPDH (Cell Signaling Technology, Danvers, MA), c-Jun N-terminal kinase (JNK), p-JNK, tumor necrosis factor receptor-associated factor 6 (TRAF6), and nuclear factor of activated T cells (NFATc1; Santa Cruz Biotechnology, Santa Cruz, CA). The gray values of the band were quantified.