Chemiluminescence reagent plus kits

Total proteins collected from the transverse colons of all subset groups (No, Lop+vehicle, Lop+GEGR) were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, after which the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was then incubated separately with the following respective primary antibody, overnight at 4℃: anti-PKC (Cell Signaling Technology Inc.), anti-p-PKC (Cell Signaling Technology Inc.), anti-Bax (Abcam, Cambridge, UK), anti-p53 (Abcam, Cambridge, UK), anti-JNK (Cell Signaling Technology Inc.), anti-p-JNK (Cell Signaling Technology Inc.) or anti-actin (Sigma-Aldrich Co.). Next, the membranes were washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, and 0.05% Tween 20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA) at a dilution of 1:1,000 for 2 h at room temperature. Finally, the membrane blots were developed using Chemiluminescence Reagent Plus kits (Pfizer, New York, NY, USA and Pharmacia, New York, NY, USA).

To investigate an alteration in the expression of ER stress marker proteins, total proteins collected from the transverse colons of No-, Lop+Vehicle and Lop+Urd treated rats were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, after which the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was subsequently separately incubated overnight at 4℃, with the following primary antibodies: eIF2α (Cell Signaling Technology Inc., Cambridge, MA, USA), p-eIF2α (Cell Signaling Technology Inc.), IRE1β (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p- IRE1β (Santa Cruz Biotechnology) and anti-β-actin (Cell Signaling Technology Inc.). The membranes were then washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, and 0.05% Tween 20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA) at a dilution of 1:1,000 at room temperature for 2 h. Finally, the membrane blots were developed using Chemiluminescence Reagent Plus kits (Pfizer, New York, NY, USA and Pharmacia, New York, NY, USA). The chemiluminescence signals that originated from specific bands were detected using FluorChemi®FC2 (Alpha Innotech Co., San Leandro, CA, USA).