Anti rabbit and anti mouse igg antibodies

K597, a DPP-4 inhibitor, and the Wako Labassay™ Glucose kit were from Wako Pure Chemical. Primary antibodies against GLUT1 and GLUT4, horseradish peroxidase-conjugated anti-goat, anti-rabbit and anti-mouse IgG antibodies and protein A/G plus-agarose were obtained from Santa Cruz Biotechnology Inc. Anti-AMPKα, anti-PI3K, anti-Akt and anti-insulin receptor (IR)β were from Cell Signaling Technology Inc. Anti-phosphotyrosine and anti-insulin receptor substrate (IRS)-1 were from Becton, Dickinson and Company. All other reagents used were of the highest grade available from commercial sources.

Proteins from IEC-6 cells or rat intestine samples were extracted using a total protein extraction kit (Biochain, Hayward, CA, USA) and quantified using the BCA protein assay kit (Pierce, Rockford, IL, USA). Proteins separated by SDS-PAGE were transferred to nitrocellulose membrane (Pierce). Membranes were blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) for 2 h at room temperature (RT). The blocked membranes were subsequently incubated with antibodies against occludin (Invitrogen, Carlsbad, CA, USA), ZO-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin and β-actin (Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The membranes were then washed and incubated with the secondary anti-rabbit and anti-mouse IgG antibodies (Santa Cruz Biotechnology) at a dilution of 1:15,000 for 1 h at RT. The protein bands were then scanned in the 800 nm channel of the Odyssey infrared imaging system (LI-COR Biosciences) and quantified with Odyssey software version 3.0 (LI-COR Biosciences).

Western blotting was performed as described previously [56 (link), 57 (link)]. The antibody against CD3ζ was purchased from Abcam. Anti-EGFR and anti-P-EGFR (Tyr1068) antibodies were purchased from Cell Signaling Technology. Anti-β-actin antibody was purchased from Sigma. Anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology.