Quantitative Analysis of Protein Expression

The total protein of the small intestinal samples was extracted via Protein Extraction Kit (Beyotime, Shanghai, China), then conventional BCA was used to measure total protein concentrations. A PAGE gel was prepared using a PAGE Gel Fast Preparation Kit (EpiZyme, Shanghai, China). After sample addition, electrophoresis was performed using PowerPac Basic (Bio-Rad, Singapore) at 80–110 V. Then, the Immobilon-P transfer membrane (Merck Millipore, Boston, US) was activated in methanol, and proteins were transferred at 200 mA for 2 h. Western Blocking Buffer (Beyotime) was used to block non-specific binding. The membrane was then incubated with primary specific polyclonal rabbit antibodies against ERK1/2, p38, PKR, and Hck (1:2,000; BIOSS, Beijing, China) or β-actin (1:300; Beyotime) at 4°C overnight. After washed using TBS-Tween three times, the membrane was subsequently incubated with secondary antibodies at 4°C for 2 h. A BeyoECL Plus P0081 88 kit were used to color the bands. After captured by FusionCapt Advance FX7 (Fusion FX; OSTC Ltd. San Diego, CA, USA), the images were processed using Image Pro-Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) to acquire quantitative data following the steps published by Wu et al. (28 (link)).