Mini trans blot system

Proteins were isolated from Caco-2 cells using RIPA buffer and subsequently subjected to Western blot analysis as described by Dinić et al. (2017) (link). Briefly, the extracted proteins (10 μg) were separated on 12% SDS–PAGE and transferred to 0.2 mm nitrocellulose membrane (GE Healthcare, Chicago, IL, United States) using Bio-Rad Mini trans-blot system (Bio-Rad, Hercules, CA, United States). The membranes were incubated for 2 h with anti-claudin (CLDN-4) antibody (1:1000; Novus Biologicals, United States) and anti-β-actin (1:1000; Thermo Fisher Scientific). The membranes were washed and incubated with appropriate HPR-conjugated secondary antibodies (goat anti-rabbit; 1:10000; Thermo Fisher Scientific) for 1 h at room temperature. Proteins were detected by enhanced chemiluminescence (Immobilon Western, Merck Millipore).

The RIPA lysate buffer (Beyotime Biotech, Nantong, China) was used to extract total proteins from the brain and the bicinchoninic acid protein assay kit (Beyotime, Shanghai, China) was used to determine the concentrations of proteins. The total proteins were run on SDS-PAGE gels and then blotted onto nitrocellulose membranes (Millipore, Billerica, MA, USA) using the BIO-RAD Mini Trans-Blot^® System (Bio-Rad, Louisville, KY, USA). The membranes were incubated with 5% skim milk to reduce nonspecific binding for 1h at room temperature. Then, the membranes were incubated overnight at 4 °C in primary antibodies. The primary antibodies used in this study were ZO-1 (1:1000; Cat#61-7300, Invitrogen), Occludin (1:500; Cat#71-1500, Invitrogen), and Claudin-5 (1:50; Cat#35-2500, Invitrogen) and GAPDH (1:1000; Cat#2118, Cell Signaling Technology). After the membranes were washed four times with TBST, they were incubated with HRP-conjugated secondary antibodies (all at 1:1000 dilution in 5% nonfat milk) at room temperature for 1 h. Then, the membranes were visualized using the enhanced chemiluminescence (ECL) reagent (Millipore, Billerica, MA, USA) and the chemiluminescence imaging system (ChemiScope 5300; Clinx Science Instruments, Shanghai, China). The band intensity was analyzed using a chemiluminescence imaging system (ChemiScope 5300; Clinx Science Instruments, Shanghai, China).

Protein lysates were obtained from a paw tissue previously pulverized in liquid nitrogen using RIPA buffer and subsequently subjected to Western blot analysis as described by Dinic et al. (2017) (link). Briefly, the extracted proteins (20 μg) were separated on 12% SDS–PAGE and transferred to 0.2 mm nitrocellulose membrane (GE Healthcare) using Bio-Rad Mini trans-blot system (Bio-Rad, Hercules, CA, United States). The membranes were incubated for 2 h with anti-myeloperoxidase (MPO) antibody (1:1000; Abcam) and anti-β-actin (1:1000; Thermo Scientific). The membranes were washed and incubated with appropriate HPR-conjugated secondary antibodies (goat anti-rabbit; 1:10000; Thermo Scientific and goat anti-mouse; 1:10000; Amersham Biosciences, Piscataway, NJ, United States) for 1 h at room temperature. Proteins were detected by enhanced chemiluminescence (Immobilon Western, Merck Millipore). The intensity of the bands was quantified using ImageJ software. The results were normalized to β-actin loading control.