Protein Extraction and Western Blot Analysis

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.