Lysis of jejunal tissue was prepared using lysis buffer (Sigma, Saint Louis, MO, USA). Total protein concentration was determined using the BCA method. Western blotting analysis was performed as described in a previous study [18 (link)]. The primary antibodies included rabbit anti-iNOS (HuaBio, Hangzhou, China), anti-ERK (Cell Signaling Technology, MA, USA), anti-phospho-ERK (Cell Signaling Technology, MA, USA), anti-p38 (Cell Signaling Technology, MA, USA), anti-phospho-p38 (Cell Signaling Technology, MA, USA), anti-JNK (Cell Signaling Technology, MA, USA), anti-phospho-JNK (Cell Signaling Technology, MA, USA), anti-NF-κB-p65 (Abcam, Cambridge, UK), anti-TRAF6 (HuaBio, Hangzhou, China), anti-HO1 (HuaBio, Hangzhou, China), and anti-GADPH (HuaBio, Hangzhou, China). The second antibody was HRP, goat anti-rabbit IgG, and goat anti-mouse IgG (HuaBio, Hangzhou, China).
Protein bands were visualized with a chemiluminescence substrate (Millipore, MA, USA) and a gel-imaging system (Tanon Science and Technology, Shanghai) and analyzed with Image-J Analysis software (NIH, Bethesda, MD, USA). As an internal control, GADPH showed no difference among the three groups. In all cases, the density values of bands were corrected after subtracting background values. GAPDH was used as an internal reference protein.
Protein bands were visualized with a chemiluminescence substrate (Millipore, MA, USA) and a gel-imaging system (Tanon Science and Technology, Shanghai) and analyzed with Image-J Analysis software (NIH, Bethesda, MD, USA). As an internal control, GADPH showed no difference among the three groups. In all cases, the density values of bands were corrected after subtracting background values. GAPDH was used as an internal reference protein.