Traf6

Tetrandrine (purity >98%) was purchased from MedChemExpress (New Jersey, USA) and was dissolved in DMSO at a 10 mmol/L stock solution and stored -20℃. Further dilution was carried out in culture medium for cells and PBS medium for animals. Primary antibodies against CTSK, CTR, MMP-9, TRAF6, TRAP, GAPDH, and β-actin were purchased from Proteintech (Wuhan, Hubei, China). Primary antibodies against NFATc1, P-PI3K, AKT, P-AKT, P50, P-P50, P65, P-P65, IκBα, P-IκBα, ERK1/2, P-ERK1/2, JNK, P-JNK, P38, and P-P38 were obtained from Cell Signaling Technologies (Beverly, MA, USA). Primary antibodies against RANKL, OPG, and the ELISA kit of RANKL were obtained from ABclonal (Wuhan, Hubei, China). The ELISA kits of OPG, IL-6, TNF-α, TRAcp5B, and CTX-I were purchased from Sangon (Shanghai, China). A CCK-8 assay kit was purchased from Dojindo (Tokyo, Japan). A leukocyte acid phosphatase staining kit was obtained from Sigma‐Aldrich (MO, USA). Recombinant M‐CSF and Recombinant m-RANKL were obtained from R&D Systems (Minneapolis, MN, USA). The cell culture medium that alpha‐modified minimal essential medium (α‐MEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Thermo Fisher Scientific (Scoresby, Vic., Australia).

Protein lysates were subjected to SDS-PAGE, and the resolved proteins were then transferred to PVDF membranes. The membranes were blocked in 5% non-fat dry milk or bovine serum albumin/fraction V (BSA, Sigma, USA) and then incubated overnight at 4 °C with the following primary antibodies: STX2 (Proteintech, USA), TRAF6 (Proteintech, USA), IKKβ (CST, USA), pIKKα/β (CST, USA), IκBα (CST, USA), pIκBα (CST, USA), P65 (CST, USA), pP65 (CST, USA) and α-Tubulin (Sigma, USA). Finally, the appropriate secondary antibodies (HRP-conjugated anti-rabbit IgG (CST, USA) and HRP-conjugated anti-mouse IgG (CST, USA) were applied. Chemiluminescent signals were detected with SuperSignal West Pico and exposed to autoradiography (HyBlot CL). α-Tubulin was used as the internal control to confirm the equal loading of the proteins.
Cell lysates for Co-IP were prepared from SW480-STX2^-Flag and LOVO. The cell lysates were pre-cleared by incubating with protein A + G Sepharose beads (Sigma, USA). Then, the individual antibodies for STX2, TRAF6, and IgG were added to the lysates, and the samples were incubated overnight at 4 °C, after which the complexes were collected with protein A + G Sepharose beads and brief centrifugation. Finally, the bound proteins were separated by SDS-PAGE.

Cells were lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing proteinase and phosphatase inhibitors. A BCA assay kit (Beyotime) was used to measure the concentration of protein. The supernatant proteins were precipitated. The protein of lytic samples was transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime, China) after separating by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime). Membranes were incubated with antibodies against gasdermin D (GSDMD, Abcam, Cambridge, UK), IL-1β (Abcam), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc-1, Abcam), TRAP (Abcam), pro caspase-1 (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-1 (Cell Signaling Technology), ASC (Cell Signaling Technology), TNF receptor-associated factor (TRAF) 2 (Proteintech, Rosemont, IL, USA), TRAF6 (Proteintech), c-Fos (Proteintech), cathepsin K (CTSK, Proteintech), matrix metalloprotein 9 (MMP9, Proteintech), and anti-actin (Beyotime) overnight at 4 °C after blocking with quick block buffer (Beyotime) for 30 min. Then, membranes were washed three times with TBS-Tween and incubated with the appropriate secondary antibody for 1 h at room temperature. The results were visualised via chemiluminescent peroxidase substrate (Proteintech).