Western Blot Protein Analysis Protocol

The protein extraction methods used in this study were adopted from our previous protocol [11 (link)]. The protein concentration in the cell extract was determined using a Bio-Rad protein assay dye reagent (Richmond, VA, USA). Aliquots of the supernatant containing 50 μg protein were separated through standard SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were incubated with anti-insulin receptor β (IRβ; 1 : 1000; Cell Signaling Technology, Beverly, MA, USA), anti-PI3-kinase p85 (1 : 1000; Cell Signaling Technology), anti-phospho-Akt (Ser473) (1 : 1000; Cell Signaling Technology), anti-Akt (1 : 500; Cell Signaling Technology, Danvers, MA, USA), antiglucose transporter (GLUT)2 (1 : 500; Millipore, Billerica, MA, USA), anti-PPARα (1 : 1000; GeneTex, Irvine, CA, USA), or anti β-actin (1 : 4000; GeneTex) antibodies at 4°C overnight. The membranes were incubated with anti-mouse IgG or anti-rabbit IgG secondary antibodies and washed thrice for 5 min each time. Protein band images were detected and captured using the UVP Biospectrum image system (Level, Cambridge, UK). Finally, all relevant protein expressions were normalized with β-actin.