Mouse anti gapdh

Rats were sacrificed under deep anesthesia; the colon tissues were promptly harvested and subjected to protein extraction using RIPA buffer and lysed. After centrifuging at 1500 relative centrifugal force (RCF) (15 min at 4°C). The concentration was determined using the BCA kit (Beyotime, China), and the rest of supernatant was stored at −80°C. Proteins were separated via 10% SDS-PAGE (Genshare Biology, China) and transferred to PVDF membranes (Millipore, Burlington, MA, United States), which were blocked in 5% non-fat milk, and incubated at 4°C overnight with either rabbit anti-TLR4 (1:1000, CST), rabbit anti-p38 (1:1000, CST), rabbit anti-pro-p38 (1:1000, CST), or mouse anti-GAPDH (1:1000, Zhongshan Golden Bridge Biotechnology) primary antibodies. The next day, the membranes were incubated with the secondary antibodies (ie, horseradish peroxidase-conjugated anti-mouse or rabbit secondary antibodies (1:5000 for both, Jackson ImmunoResearch, USA)). The visualization was via the Clarity Western ECL Substrate (EMD Millipore) and the quantification of band intensities was via the Fiji ImageJ software.