Af5314

The total protein was extracted from ilea and then quantitated using BCA Protein Assay kit. Equal amounts of protein (20 μg) from different samples were separated by 6∼15% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk for 4 h at room temperature, then probed with ZO-1 (1:1,000, AF5145, Affinity), claudin-1 (1:1,000, AF0127, Affinity), occludin (1:1,000, DF7504, Affinity), p65 (1:1,000, AF5002, Affinity), MLCK (1:1,000, AF5314, Affinity), MLC2 (1:1,000, 36725, CST), and β-actin (1:3,000, ab8226, Abcam) was used as loading control, overnight at 4°C and with secondary antibody at RT for 1 h. The images were captured using ChemiDoc MP imaging system (Bio-Rad).

Cellular samples were collected and lysed, and the protein concentration was determined using the BCA method. Based on the target protein molecular weight, a suitable concentration of protein electrophoresis gel was selected for protein electrophoresis, membrane transfer, and then blocked for 1 h. Primary antibodies against ZO-1 (1:1000, Cat#AF5145, Affinity Biosciences, China), MLCK (1:800, Cat#AF5314, Affinity Biosciences, China), p-MLC (1:1000, Cat#95777, Cell Signaling Technology, USA), MLC (1:1000, Cat#8505, Cell Signaling Technology, USA), and β-actin (1:2000, Cat#4970, Cell Signaling Technology, USA) were incubated overnight at 4° C on a shaker. Secondary antibodies (1:5000, Cat#58802, Cell Signaling Technology, USA) were then incubated for exposure, and the images were recorded using a Bio-Rad Chemidoc XRS+ imaging system. The protein expression levels were calculated using the software ImageJ for grayscale analysis.