Claudin 4

16HBE cells were seeded onto poly-L-lysine-coated coverslips (Solarbio, China) and treated as previously described. At the indicated time, the cells were washed in PBS, fixed with 4% PFA (Solarbio, China) and permeabilized with 1% Triton X-100 in PBS. The cells were blocked with 5% BSA at room temperature for 1 h and then incubated with the primary antibodies in blocking solution against EV-A71/CV-A16-VP1 (1:1,000, Millipore, USA), Nectin1 (1:100, Novusbio, USA), Claudin4 (1:200, Abcam, USA), E-cadherin (1:500, Abcam, USA) and ZO-1 (1:200, Abcam, USA) overnight at 4°C. Next, cells were washed with PBS three times and then incubated with Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Abcam, USA), Alexa Fluor 647-conjugated donkey anti-mouse IgG (Millipore, USA) or Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Biolegend, USA) for 1 h at room temperature. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, 1:4,000, Beyotime, China). Slides were mounted with antifade reagent (Solarbio, China) and observed using a laser-scanning confocal microscope (Leica, Germany). The images were captured and processed using Adobe Photoshop 7.0 software.

Minimum Essential Medium (MEM) with Earle’s Balanced Salts, fetal bovine serum (FBS), nonessential amino acid (NEAA), antibiotics (penicillin/streptomycin solution), and sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid) were obtained from Thermo Fisher Scientific (Logan, UT, United States). Twelve-well Transwell and Hanks’ Balanced Salt Solution (HBSS) were purchased from Welgene (Gyeongsan, Gyeongsangbuk-do, Republic of Korea). Antibodies against p65, p-p65, IKKα/β, p-IKKα/β, IκBα, p-IκBα, MLCK, p-MLC, E-cadherin, MKK4/7, p-MKK4/7, P38 MAPK, p-P38 MAPK. SAPK/JNK, p-SAPK/JNK, ERK, p-ERK, and α/β-Tubulin were purchased from Cell Signaling Technology (Danvers, MA, United States). Antibodies against lamin B1, ZO-1, occludin, claudin-4, claudin-2, ATF-2, and p-ATF2 were purchased from Abcam (Cambridge, Cambridgeshire, United Kingdom). The primary antibody against β-actin was purchased from Santa Cruz Biotech (Santa Cruz, CA, United States). Lipopolysaccharide (LPS) derived from Samonella enterica serotype typhimurium was purchased from Sigma-Aldrich (St. Louis, MO, United States), and tumor necrosis factor (TNF)-α was purchased from NKMAX (Seongnam, Gyeonggi-do, Republic of Korea).

Cell lysates were prepared with RIPA buffer supplemented with protease inhibitor cocktail (Beyotime, Shanghai, China). An equal amount of protein from each sample was loaded onto 10% or 15% SDS-PAGE gel and then blotted onto nitrocellulose membranes. The membranes were blocked with 5% skim milk and then probed with primary antibodies at 4 °C overnight. After incubation with horseradish peroxidase-labeled secondary antibody (Beyotime) for 1 h at room temperature, the membranes were developed with enhanced chemiluminescence reagents (Thermo Fisher). The sources of antibodies were as follows: antibodies against CXCL13, CXCR5, ZO-1, occludin, and claudin-4 were purchased from Abcam; antibodies against p38, phosphorylated p38 (p-p38), and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA). GAPDH levels were used as the loading control.

The ipsilateral cortex around the infarct area and the colon tissue were collected and stored at −80°C until further use. The brain tissues were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China) containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Beyotime Biotechnology, China) using a handheld homogenizer and incubated on ice for 20 min. The lysates were centrifuged at 12,000 × g for 20 min at 4°C, and the supernatants were transferred to fresh tubes. The protein samples were resolved on a 12% or 10% SDS-PAGE gel and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat milk at room temperature for 1 h and then incubated with primary antibodies against ZO-1 (1:1,000; Abcam), occludin (1:1,000; Abcam), claudin-4 (1: 1,000; Abcam), ICAM-1 (1:1,000; Abcam), VCAM-1 (1:1,000; Abcam), MMP-9 (1:1,000; Abcam), and β-actin (1:5,000; Abcam) at 4°C overnight. The membrane was washed with Tris-buffered saline (TBS) containing 0.1% Tween 20 and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody. The membrane was then visualized with an enhanced chemiluminescence system (Thermo Scientific, Rockford, IL). The band intensity was assessed using Image Lab software.

The ipsilateral cortex around the infarct area and the colon tissue were collected and stored at −80°C until further use. The brain tissues were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China) containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Beyotime Biotechnology, China) using a handheld homogenizer and incubated on ice for 20 min. The lysates were centrifuged at 12,000 × g for 20 min at 4°C, and the supernatants were transferred to fresh tubes. The protein samples were resolved on a 12% or 10% SDS-PAGE gel and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat milk at room temperature for 1 h and then incubated with primary antibodies against ZO-1 (1:1,000; Abcam), occludin (1:1,000; Abcam), claudin-4 (1: 1,000; Abcam), ICAM-1 (1:1,000; Abcam), VCAM-1 (1:1,000; Abcam), MMP-9 (1:1,000; Abcam), and β-actin (1:5,000; Abcam) at 4°C overnight. The membrane was washed with Tris-buffered saline (TBS) containing 0.1% Tween 20 and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody. The membrane was then visualized with an enhanced chemiluminescence system (Thermo Scientific, Rockford, IL). The band intensity was assessed using Image Lab software.