Anti occludin

Cells grown on filter supports were rinsed twice with PBS and fixed with 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, United States) for 30 min at room temperature. After permeabilization for 7 min with 0.5% Triton X-100 (Sigma Aldrich, St. Louis, MO, United States), cells were blocked 10 min at room temperature with blocking solution containing 5% goat serum (Gibson, Carlsbad, CA, United States), 0.05% Triton X-100, and 1% bovine serum albumin (BSA; Sigma Aldrich, St. Louis, MO, United States). Afterwards, cells were incubated for 1 h at 37°C with primary antibodies anti-occludin (1:100; Sigma Aldrich, St. Louis, MO, United States), anti-claudin-5 (1:100; Invitrogen, Carlsbad, CA, United States), and anti-zonula occludens protein-1 (ZO-1, 1:100; BD Biosciences, Franklin Lakes, NJ, United States) followed by the secondary anti-rabbit or anti-mouse antibody conjugated to Alexa-Fluor 488 or 594 (1:500; Invitrogen, Carlsbad, CA, United States) for 1 h at 37°C. For apoptosis detection, cells were stained with the TUNEL kit (In situ Cell Death Detection Kit, Roche AG, Mannheim, Germany) according to manufacturer’s instructions. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; 1:1000; Roche AG, Basel, Switzerland). Visualization was performed by confocal laser-scanning microscopy (CLSM; Zeiss LSM780, Jena, Germany).

The cells were scraped using ice-cold RIPA buffer in the presence of a protease and phosphatase inhibitor cocktail (10 μg/mL; Thermo Fisher Scientific, Waltham, MA, USA) and sonicated three times. Following incubation on ice for 30 min, the samples were centrifuged at 10,000× g for 20 min, and the supernatants were recovered and mixed with 2× Laemmli sample buffer and 2-mercaptoethanol (20%).
The Bradford colorimetric assay was used to determine the protein concentration and equal amounts of cell proteins (30 μg/lane) were separated by SDS-PAGE (10–12% polyacrylamide gel) and then electro-transferred onto nitrocellulose membranes by a Trans-Blot Turbo (Mini Nitrocellulose, Bio-Rad, Hercules, CA, USA). Incubation was performed overnight at 4 °C with primary antibodies: rabbit polyclonal anti-occludin (Sigma Aldrich) and mouse monoclonal anti-actin (Santa-Cruz Biotechnologies, Dallas, TX, USA). HRP-conjugated secondary antibodies (1:3000; Bio-Rad, CA, USA) were incubated with the membranes for 1 h. The blots were then detected using an enhanced chemiluminescence substrate (ECL Substrates, Bio-Rad, CA, USA). Densitometric analysis was performed using Image Lab software 6.1.0. The results were expressed as arbitrary units (A.U.) and represented as the mean of three independent experiments.

Initially, the lysis buffer (Beyotime, Shanghai, China) was added to the collected cells to conduct 30 min of homogenization on ice. Furthermore, the lysed cells were subjected to 30 min of centrifugation under 4°C to obtain supernatant. Then, the Bradford method was adopted to measure the total protein content. 20 μg of protein was separated by 10% SDS-PAGE, followed by transfer onto PVDF membranes. Subsequently, Tris-buffered saline containing 3% skimmed milk was employed to incubate membranes along with suitable primary antibodies under 4°C overnight, including anti-EGFR, anti-AKT3, anti-JAM, anti-β-actin, or anti-occludin antibodies (Sigma, St. Louis, USA). After washes, the membranes were incubated with HRP-conjugated goat anti-rat IgG (Sigma). Then, an ECL detection kit (Bio-Rad, Hercules, CA, USA) was applied to detect binding antibodies through chemiluminescence staining. Gel Doc XR system (Bio-Rad, Hercules, CA, USA) was adopted for quantifying the band density, and the protein expression changes were calculated relative to control.

The cells were scraped using ice-cold RIPA buffer in the presence of a protease and phosphatase inhibitor cocktail (10 μg/mL; Thermo Fisher Scientific, Waltham, MA, USA) and sonicated three times. Following incubation on ice for 30 min, the samples were centrifuged at 10,000× g for 20 min, and the supernatants were recovered and mixed with 2× Laemmli sample buffer and 2-mercaptoethanol (20%).
The Bradford colorimetric assay was used to determine the protein concentration and equal amounts of cell proteins (30 μg/lane) were separated by SDS-PAGE (10–12% polyacrylamide gel) and then electro-transferred onto nitrocellulose membranes by a Trans-Blot Turbo (Mini Nitrocellulose, Bio-Rad, Hercules, CA, USA). Incubation was performed overnight at 4 °C with primary antibodies: rabbit polyclonal anti-occludin (Sigma Aldrich) and mouse monoclonal anti-actin (Santa-Cruz Biotechnologies, Dallas, TX, USA). HRP-conjugated secondary antibodies (1:3000; Bio-Rad, CA, USA) were incubated with the membranes for 1 h. The blots were then detected using an enhanced chemiluminescence substrate (ECL Substrates, Bio-Rad, CA, USA). Densitometric analysis was performed using Image Lab software 6.1.0. The results were expressed as arbitrary units (A.U.) and represented as the mean of three independent experiments.

Total protein was extracted from Caco-2 cells using lysis buffer, and protein concentrations were adjusted to a known concentration using Bradford reagent (Sigma, St. Louis, MO, USA), before conducting electrophoresis. TJ proteins were resolved on 8% (for occludin and ZO-1) and 15% (for Claudin-4) SDS–polyacrylamide mini-gels using a Mini-PROTEAN^® II Multiscreen Apparatus (Bio-Rad Laboratories, Hercules, CA, USA), and then transferred to nitrocellulose membranes (0.2 μm; Bio-Rad). Non-specific binding was ensured using 5% BSA, and membranes were incubated with anti-human primary antibodies, including anti-Claudin 4, anti-occludin, anti-ZO-1, and anti-GAPDH (Sigma, St. Louis, MO, USA) at 4 °C overnight in the dark. Membranes were washed three times with Tris-buffered Saline-Tween (TBST) and incubated with secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA), for 60 min. The membranes were washed three times with TBST, reacted against a Western ECL substrate (Bio-Rad Laboratories (Canada) Ltd., Mississauga, ON, Canada) for 5 min, and analyzed using the ChemiDoc^TM MP Imaging System (Bio-Rad Laboratories Ltd., Hercules, CA, USA).