Phospho iκbα

Cells were scraped in 1× lysis buffer from Cell Signaling Technology (catalog no. 9803) with protease and phosphatase inhibitors and 0.5% SDS, followed by sonication with F60 Sonic Dismembrator (Fisher Scientific). The amount of protein in each sample was quantified using Pierce BCA Protein Assay Kit. All samples were diluted to the same concentration. For western blot analysis, samples were boiled at 95 °C for 5 min and 20 μg of total protein was loaded on Criterion™ TGX™ precast gels (BioRad Laboratories). Gels were run for 1 h at 150 V followed by transferring proteins onto a nitrocellulose membrane using an iBlot system (Invitrogen). Five percent non-fat dried milk in tris-buffered saline, tween 20 was used to block the membranes for 1 h. After blocking, primary antibodies against GCLC (Abcam), NQO1 (Abcam), HO1 (Abcam), TXNRD1 (Abcam), ACTB (MP Biomedical), Phospho-IκBα (Abcam), and GAPDH (Abcam) were incubated overnight at 4 °C, followed by washing with 1× tris-buffered saline, tween 20 for 30 min. Secondary antibodies were then incubated at room temperature for 45 min. After washing the membranes for 30 min, Super Signal West Femto Substrate was added to detect horseradish peroxidase on the membranes. Chemiluminescence was measured and quantified using the Syngene gel imaging system.

Chrysin (purity > 98% Figure 1A) was purchased from Aladdin reagent co., LTD (Shanghai, China). Recombinant mice M-CSF was obtained from R&D Systems (Minneapolis, MN, United States). The purification process of Recombinant RANKL followed the protocol as previously reported (Xu et al., 2000 (link)). The CCK8 assay kits were purchased from Promega (Madison, WI, United States). Antibodies for ERK, phospho-ERK, IκBα, phospho-IκBα, and β-Actin were obtained from Abcam (Cambridge, United Kingdom). Anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, anti-phospho-NF-κB p65, and anti-NF-κB p65 were obtained from Cell Signaling Technology (Boston, United States).