Anti il 6

The proteins in the sample were harvested as described above. The experiment procedure was also following our previous publication [18 (link)]. The antibodies used were listed as below: anti-JAK1, anti-JAK2, anti-STAT1, anti-pSTAT1, anti-STAT3, anti-pSTAT3 (Cell Signaling, Beverly, MA, USA; catalog number:#3344, #3230, #9172, #7649, #9139, #9136); anti-SOCS3 (GeneTex, Irvine, CA, USA; catalog number:GTX23693); anti-IFN-γ (Abcam, Cambridge, UK; catalog number:ab9657); anti-TLR-4 (Proteintech, Rosemont, IL, USA; catalog number:19811-1-AP); anti-IL-6 (Bioworld Technology, Bloomington, MN, USA; catalog number:BS6419); at room temperature (RT) for 1 h. The protein bands on the membrane were detected with an ECL-Plus Western Blot Detection system (GE Healthcare UK LTD) according to the instructions of the manufacturer. All experiments were replicated at least thrice.

RAW264.7 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). WKYMVm and WRWWWW (WRW4) with purity > 95% was synthesized by GL Biochem and was dissolved in acetonitrile to prepare stock concentrations of 0.1, 1, 2, 5 and 10 μmol/L. Dulbecco's Modified Eagle's Medium (DMEM), alpha minimum essential medium (α‐MEM) and foetal bovine serum (FBS) were purchased from Invitrogen‐Gibco. RANKL and soluble mouse recombinant M‐CSF were obtained from R&D Systems. The Acid Phosphatase, Leukocyte (TRAP) assay kit and Actin Cytoskeleton and Focal Adhesion (FAK) Staining Kit were purchased from Sigma (Sigma‐Aldrich) and Merck, respectively. The Cell Counting Kit‐8 (CCK‐8) was purchased from Beyotime Biotechnology. Anti‐phospho‐NF‐kBp65, anti‐NF‐kBp65, anti‐STAT3, anti‐phospho‐STAT3 (Ser727), anti‐CTSK, anti‐c‐Fos, anti‐NFATc1, anti‐CD9, anti‐gp130, anti‐IL6 and anti‐ß‐actin antibodies were purchased from Bioworld Technology. Mouse IL‐1β, IL‐6 and TNF‐α ELISA kits were procured from Novus.

The proteins in the sample were harvested using RIPA lysis buffer (Merck Millipore, Burlington, MA, USA). The experimental procedure was also following our previous publication [24 (link)]. The antibodies used are listed as follows: anti-JAK1 (BD biosciences, East Rutherford, NJ, USA); anti-JAK2, anti-STAT1 (Cell Signaling, Beverly, MA, USA); anti-pSTAT1, anti-STAT3 (Cell Signaling, Beverly, MA, USA); anti-pSTAT3, anti-SOCS3 (Abcam, Cambridge, UK); anti-insulin receptor (Abcam, Cambridge, UK); anti-TLR-4 (Proteintech, Chicago, IL, USA); anti-IL-6 (Bioworld Technology, St Louis Park, MN, USA); and anti-β-actin (Santa Cruz, Santa Cruz, CA, USA) at room temperature (RT) for 1 h. After the incubation with the appropriate secondary horseradish peroxidase-conjugated IgG antibody (R&D Systems) for 30 min at RT, the protein bands on the membrane were detected with ECL-Plus Western Blot Detection system (GE Healthcare UK LTD) according to the instructions of the manufacturer. All experiments were replicated at least thrice.