2.10. Western Blot Analysis

The ipsilateral cortical tissues, gastrocnemius muscles, liver tissues, and bEnd.3 cell lysates were homogenized using a Tissue Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of extracted proteins were separated through a standardized SDS-PAGE (8% and 12%) and transferred onto PVDF membranes, which were sequentially incubated with 5% skim milk, corresponding with primary antibodies, IgG-HRPs, and enhanced chemiluminescence Western blotting reagents (n = 8 per group). The chemiluminescence on the membranes were visualized using a G:BOX mini multi fluorescence and chemiluminescence imaging system (Syngene, Frederick, MD, USA) and quantified by Image J software (National Institute of Health, Bethesda, MD, USA). Primary antibodies were recognized, which included Receptor-Interacting Protein Kinase 1 (RIPK1, 1:1000), Microtubule-Associated Protein 2 (MAP-2, 1:1000), Cluster of Differentiation 68 (CD68, 1:1000), Glial Fibrillary Acidic Protein (GFAP, 1:1000), Ubiquitin Protein Ligase E3 Component N-Recognin 1 (Ubr1, 1:1000), Tumor Necrosis Factor-α Receptor Type I (TNFRI, 1:1000), IKK-α/β (1:1000), p65 (1:1000), phospho-p65 (Serine-536, 1:500), Stat3 (1:1000), phospho-Stat3 (Tyrosine-705, 1:500), Zonula Occludens-1 (ZO-1, 1:1000), c-Jun N-terminal Kinase (JNK, 1:1000), phospho-JNK (Threonine-183/Tyrosine-185, 1:500), Akt (1:1000), phospho-Akt (Serine-473, 1:500), Janus Kinase 2 (Jak2, 1:1000), phospho-Jak2 (Tyrosine-1007, 1:1000), Insulin Receptor Substrate-1 (IRS1, 1:1000), phospho-IRS1 (Serine-307, 1;500), Suppressors of Cytokine Signaling 3 (SOCS3, 1:1000), Synaptosome Associated Protein 25 (SNAP25, 1:1000), Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH, 1:3000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-IKK-α/β (Serine-176/180, 1:500), and phospho-IRS1 (Tyrosine-895, 1:500) (Cell Signaling, Beverly, MA, USA).