Horseradish peroxidase conjugated anti mouse igg

Proteins were extracted from frozen intestinal mucosae by grinding with RIPA lysis buffer and phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China). Protein concentrations were measured using a bicinchoninic acid (BCA) kit (Beyotime). Thereafter, 40 μg of protein/lane was electrophoresed in 12% SDS–PAGE gels, followed by transfer to polyvinylidene difuoride membranes and blocking with 5% non-fat dry milk in Tris-buffered saline Tween-20 buffer (0.05% Tween-20, 100 mmol/L Tris–HCl, and 150 mmol/L NaCl, pH 8.0) for 2 h. After blocking, the membranes were incubated overnight with primary antibodies at 4℃. The primary antibodies were Toll-like receptor 4 (1:1000; Abcam; Cambridge, MA, USA) and β-actin (1:5000; Proteintech; Rosemont, IL, USA). The membranes were washed in TBST three times and were processed with an HRP-conjugated secondary antibody (horseradish peroxidase-conjugated anti-mouse IgG, 1:10000; Proteintech) for 60 min at room temperature. The blots were developed using an enhanced chemiluminescence reagents (Merck Millipore, Darmstadt, Germany) followed by autoradiography. Images were recorded using a Luminescent Image Analyzer LAS-4000 system (Fujifilm, Tokyo, Japan) and were quantified by Image-Pro Plus 6.0. β-actin was used as the internal standard to normalize the signals.

Total RNA was extracted from cells using Trizol (Takara) as per the guidelines provided by the manufacturer. Following that, mRNA was purified by using Sigma-Aldrich’s GenElute Messenger RNA (mRNA) Miniprep Kit (Sigma). The same quantities of mRNA that were diluted in a series were placed onto a nylon membrane (Biosharp) and then exposed to ultraviolet light after being denatured at 70 °C for 5 min. To prevent interference, a block was carried out at room temperature for a duration of 1 hour using 5% milk in PBST. Subsequently, the anti-m¹A antibody (D345-3, MBL; 1:1000) was incubated overnight. Following PBST washed three times for five minutes, a diluted (1:5000) horseradish peroxidase-conjugated antimouse IgG (Proteintech, Wuhan) was incubated at room temperature for an hour with the membranes. A further three times of washing with PBST were performed for five minutes each, followed by signal detection using ECL reagents (Boster, Wuhan).

Collected fresh samples were homogenized in RIPA lysis buffer with a protease inhibitor. The homogenate was centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatants were collected immediately. Protein samples were separated by SDS-PAGE gels and transferred onto PVDF membranes (Millipore, United States). The membranes were blocked with 5% BSA for 2 h at room temperature and then probed with primary antibodies overnight with gentle shaking at 4°C. After three washes in PBS-T, the membranes were subsequently incubated with horseradish peroxidase–conjugated anti-mouse IgG (1:5000, Proteintech, United States) or anti-rabbit IgG (1:6000, Proteintech, United States) secondary antibodies for 2 h at room temperature. Membranes were washed again, and the protein bands were visualized using enhanced chemiluminescence (ECL). The exposed films were scanned and analyzed by Quantity One analysis software.
The primary antibodies used were the following: rabbit anti-NIK (1:200, Abcam, United Kingdom); rabbit anti-IKKα (1:1000, Cell Signaling Technology, United States); rabbit anti-NF-κB p100/52 (1:1000, Cell Signaling Technology, United States); mouse anti-β-actin (1:5000, proteintech, United States); rabbit anti-Histone H3 (1:1000, Cell Signaling Technology, United States); rabbit anti-TNF-α (1 ug/ml, Abcam, United Kingdom); and rabbit anti-IL-β (0.2 ug/ml, Abcam, United Kingdom).