Anti nf κb p65

For immunoblotting, the intestinal tissues were lysed on ice and centrifuged at 4 °C and at 12,000 g for 10 min. The supernatant was extracted using RIPA buffer (Absin, Shanghai, China) containing proteinase inhibitor cocktail (Innovation, USA). The concentration of protein was then determined using a BCA kit (Beyotime Institute of Biotechnology). 40 μg protein/lane was separated by 10% SDS-PAGE. The separated proteins were subsequently transferred onto polyvinylidene fluoride membranes (EMD Millipore) and blocked with 5% skimmed milk for 1.5 h. The membrane was incubated with anti-TRPV1 (Proteintech, Chicago, USA), anti-TRPV4 (Abcam, Cambridge, UK), anti-NF-κB p65 (Bioss, Beijing, China), anti-5-HTR3A (Bioss, Beijing, China) and anti-iNOS (Bioss, Beijing, China) antibodies overnight at 4 °C, followed by incubation with secondary antibodies (Abcam, Cambridge, MA, USA) for 1 h at room temperature. Protein bands were scanned and visualized using an enhanced chemiluminescence detection system (EMD Millipore). Each band was quantified via Image J software (National Institutes of Health). The gray value of the target protein was normalized to that of GAPDH.

After growth and challenge utilizing a 6-well format (CELLTER, Santiago, Chile), lysis of the cells was accomplished with the RIPA buffer (Solarbio, Beijing, China), followed by electrophoretic shift of the protein samples onto the PVDF (polyvinylidene difluoride) membranes (Solarbio, Beijing, China). The next step was a 1-h blockage of unbound PVDF sites with buffer and subsequent incubation based on optimal primary antibody dilutions, such as anti-phospho-NF-κB p65 & anti-NF-κB p65 (both Bioss, Beijing, China), anti-phospho-IκB alpha (Bioss, Beijing, China), anti-phospho-p38 MAPK & anti-phospho-ERK1/2 MAPK (both Cell Signaling Technology, MA, USA), anti-beta-Actin (Bioss, Beijing, China), anti-phospho-NF-κB p65 (Bioss, Beijing, China), anti-phospho-JNK1 (Bioss, Beijing, China), and anti-GAPDH (Proteintech, Wuhan, China) antibodies, for the proteins of interests for 12 h at 4°C. After rinsing four times with PBS-containing Tween 20 (PBST), a further 1-h incubation of the membranes proceeded with alkaline phosphatase (AP)-labeled secondary antibodies at 37°C. Then, the membranes were rinsed four times in PBST (Solarbio, BeiJing, China). A BCIP/NBT Substrate color kit was used for visualization.