Superblock blocking buffer in tbs

Briefly, protein extraction was performed using a lysis cocktail, including a radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA), 0.5M EDTA (Thermo Fisher Scientific, Waltham, MA, USA) and a protease inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) cocktail. The lysed tissues were then homogenized. Jejunum and ileum samples were vortexed, followed by centrifugation at 13,000× g. Then, 15 µg of the protein samples was electrophoresed in 10% SDS-PAGE, and then electro transferred onto nitrocellulose membranes (Bio-Rad). The membranes were then blocked in Superblock blocking buffer in TBS (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were incubated with anti-claudin-2, -3, and -4 (Thermo Fisher Scientific, Massachusetts, USA) at 4 °C, followed by washing with PBS/Tween prior to the incubation with a secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature. Antibody binding was detected using a Supersignal West Pico chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA).

The cells were seeded in 35 mm culture dishes at a density of 2 × 10⁵ cells/well and incubated with 50 µg/mL of tunicamycin for designated periods of time. After the designated treatment, cells were lysed in 300 µL of sodium dodecyl sulfate (SDS) buffer. The samples (10 µL) were subjected to electrophoresis on 4–12% or 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE; Invitrogen) for 90–120 min at 20 mA and then transferred onto polyvinylidene difluoride (PVDF) membranes (Invitrogen) using iBlot (Life Technologies, Carlsbad, CA, USA). The membranes were blocked in Super Block Blocking Buffer in TBS (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h and incubated overnight at 4 °C in the presence of primary antibodies at dilutions of 1:1000–1:3000 in Tris Buffered Saline with Tween 20 (TBS-T). After three washes with TBS-T, the membranes were incubated with the corresponding species-appropriate secondary antibodies at dilutions of 1:1000–1:3000 in TBS-T for 1 h. Then, the immunoreactive bands on the membrane were visualized with ECL Prime Western Blotting Detection Reagents (GE healthcare, Chicago, IL, USA) and the images were captured using the LAS-4000 mini (FUJIFILM, Tokyo, Japan). The band density was analyzed using Multi Gauge version 3.1 (FUJIFILM). The quantitative densitometric values for each protein were normalized to β-actin.