Multiplex bead assay for detecting cytokines and phosphorylated proteins of the signaling pathway
Multiplex bead assays were used to quantify the amounts of IL-10, IL-13, IL-12p70, IL-4, IL-6, IL-12, IL-18, IL-1α, IL-1β, monocyte chemoattractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein (MIP)-1α, MIP-2, and TNFα (the MILLIPLEX MAP Rat Cytokine/Chemokine Magnetic Bead Panel; Cat # RECYTMAG-65K-12, Billerica, MA, USA) and P-CREB, P-JNK, P-NF-κB, P-P38MAPK, P-ERK1/2, P-Akt, P-p70S6K, P-STAT3 and P-STAT5 (MILLIPLEX® MAP 9-Plex Multi-Pathway Signaling Magnetic Bead Kit for Phosphoprotein, 96-well; Cat # 48-680MAG, Billerica, MA, USA) in liver protein extracts. All assays were performed and analyzed according to the manufacturer’s instructions. The plate was analyzed using a Luminex 200™ analyzer and xPONENT (Billerica, MA, USA) software, and the median fluorescent intensity (MFI) data were saved and analyzed using a five-parameter logistic or spline curve-fitting method to calculate the analytical concentrations in the samples.