Cst 4695

Twenty grams of mouse intestinal tissue was weighed, washed with cold PBS 2–3 times, and cut into small pieces. Then, 250 μL of RIPA lysis solution was added, and the sample was ground in a tissue-grinding apparatus. The homogenate was transferred to a centrifuge tube, shaken, and centrifuged at 12 000×g for 5 min, after which the supernatant was collected as the total protein solution. A BCA protein assay kit (Beyotime Biotechnology, Shanghai) was used to quantify the extracted proteins. After separation by SDS‒PAGE, the proteins were transferred to PVDF membranes. The PVDF membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20. The membranes were then incubated with primary antibodies against Muc-2, Occ, Cla, ERK1/2 (CST-4695, Cell Signaling Technology, USA), and p-ERK1/2 (CST-4370, Cell Signaling Technology, USA). The cells were incubated overnight at 4 °C and then incubated with the secondary antibody for 2 h at 37 °C. Protein bands were detected using enhanced chemiluminescence and imaged using the Clinx Chemiluminescence Imaging System. The intensity of the β-actin protein band was used as an internal reference.

Cells were homogenized in 100 μL Kaplan buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% NP-40, 10% glycerol, and a protease inhibitor cocktail (Roche Diagnostics)). The lysate was subject to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and WB analysis with a standard procedure [62 (link)] using antibodies against AKT (1:1000; CST 4691; Cell Signaling Technology, Beverly, MA, USA), phospho-AKT (1:1000; CST 4056; Cell Signaling Technology), ERK1/2 (1:1000; CST 4695; Cell Signaling Technology), phospho-ERK1/2 (1:1000; CST 9101; Cell Signaling Technology), p38 (1:1000; CST 9212; Cell Signaling Technology), phospho-p38 (1:1000; CST 9215; Cell Signaling Technology), JNK (1:1000; CST 9252; Cell Signaling Technology), phospho-JNK (1:1000; CST 4668; Cell Signaling Technology), c-JUN (1:1000; CST 9165; Cell Signaling Technology), phospho-c-JUN (1:1000; CST 3270; Cell Signaling Technology), MBP (1:1000; M3821; Sigma-Aldrich), MAG (1:1000; MAB1567; Chemicon, Temecula, CA, USA), P0 (1:1000; ab31851; Abcam, Cambridge, UK), HIF1α (1:1000; CST 14179; Cell Signaling Technology), and β-actin (1:1000; CST 4970; Cell Signaling Technology). Protein expression levels were determined using the MF-ChemiBIS 3.2 imaging system (Berthold Technologies, Bad Wildbad, Germany).