Western Blot Analysis of Cell Signaling Proteins

WB analysis was performed as previously described.^{51 (link)} Cells were lysed using the M-PER Mammalian Protein Extraction Reagent (ThERMO Scientific), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed. The following antibodies were used: HMGB1 (1:1000, ab79823, Abcam), HSP70 (1:1000, ab181606, Abcam), Calreticulin (1:1000, ab92516, Abcam), RAGE (1:1000, ab216329, Abcam), phosphorylated NF-κB [1:1000, 3033, Cell Signaling Technology (CST)], NF-κB (1:1000, 8242, CST), phosphorylated Akt (1:1000, 4060S, CST), Akt (1:1000, 4691S, CST), phospho-MAPK Family Antibody Kit (1:1000, 9910, CST), a MAPK Family Antibody Kit (1:1000, 9926, CST), β-Actin (1:1000, 4970S, CST) and GAPDH (1:1000, 60004-1-Ig; Proteintech). Proteins were visualized by using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd).Images were prepared using ChemiDoc XRS + system (BioRad, China) and quantification analyses were performed by Bio-Rad Image Lab software (BioRad, China).