Protein Extraction and Western Blotting

The total protein content was obtained from the colon tissues of the C57BL/6 mice by applying Pro-Prep Protein Extraction Solution (Intron Biotechnology Inc., Seongnam, Republic of Korea) in accordance with the manufacturer’s protocol. After centrifugation at 13,000 rpm/min for 5 min, the protein concentrations were determined using a SMART™ Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific Inc.). Proteins were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h and then transferred to nitrocellulose membranes at 40 V for 2 h. Membranes were then incubated at 4 °C with the following primary antibodies overnight: anti-p38 (Cell Signaling Technology Inc., Cambridge, MA, USA), anti-p-p38 (Cell Signaling Technology Inc.), anti-Protein kinase C (PKC) (Cell Signaling Technology Inc.), anti-phospho-PKC (p-PKC) (Cell Signaling Technology Inc.), anti- Akt serine/threonine kinase (AKT) (Cell Signaling Technology), anti-p-AKT (Cell Signaling Technology), anti-Extracellular signal-regulated kinase (ERK) (Cell Signaling Technology Inc.), anti-p-ERK (Cell Signaling Technology Inc.), anti-PI3K (Cell Signaling Technology), anti-p-PI3K (Cell Signaling Technology), anti- c-Jun N-terminal kinases (JNK) (Cell Signaling Technology Inc.), anti-p-JNK (Cell Signaling Technology Inc.), or anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Cell Signaling Technology Inc.). The membranes were then washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 0.05% Tween 20) and incubated with 1:2000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Invitrogen) at room temperature for 1 h. Blots were developed using Amersham ECL Select Western Blotting detection reagent (GE Healthcare, Little Chalfont, UK). Chemiluminescence signals from specific bands were detected using FluorChemi^®FC2 (Alpha Innotech Co., San Leandro, CA, USA).