Bca assay kit

According to the current protocol, the colon tissues were homogenized in RIPA lysis buffer. Subsequently, the samples were normalized to the same protein concentration using a BCA assay kit (Solarbio, Beijing, China). Protein samples (30 µg) were separated by electrophoresis on a 15% SDS–polyacrylamide gel and then transferred onto a PVDF membrane. After blocking with 5% skim milk, the membrane was incubated overnight at 4 °C with the primary antibodies, followed by a 60 min incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. The expression of ZO-1, Occludin, Claudin-3, p38, p-p38, JNK, p-JNK, ERK, p-ERK, NF-κB p65, p-p65, IκBα, p-IκBβ, and GADPH was detected. The protein blot bands were quantitatively analyzed using ImageJ software 6.0 [11 (link)].

Cell pellets were resuspended in RIPA lysis buffer (50 mM Tris–HCl pH 7.4, 1 mM EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 0.1% SDS) and incubated on ice for 30 min to facilitate protein extraction. Following centrifugation at 12,000 × g for 10 min at 4 °C, the soluble protein fraction was collected from the supernatant. Protein quantification was performed using the BCA assay kit (Solarbio, Beijing, China, Cat#PC0020). Subsequently, 20–50 μg of total protein was denatured, resolved by SDS-PAGE, and transferred onto PVDF membranes. Membranes were blocked for 1 h at room temperature (RT) and then incubated with the appropriate primary antibody overnight at 4 °C, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at RT. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection reagent and captured with the Amersham Imager 800 (GE Healthcare Life Sciences, Pittsburgh, PA, USA).