Ab246504

The total protein was extracted from mouse gastric mucosa tissues or cells using radio immunoprecipitation assay lysis (Beyotime). The protein concentration was estimated using BCA Kit (20201ES76, Yeasen Company, Shanghai, China). Subsequent to separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, the protein was electrotransferred onto a polyvinylidene fluoride membrane (IPVH85R, Millipore, Germany). Subsequent to 1-h 5% bovine serum albumin blocking, the membrane received overnight probing at 4 °C with the primary antibodies to AQP5 (PA5-97290, 1:1000, Invitrogen), β-catenin (Ab32572, 1:1000, Abcam), p-β-catenin (Ab246504, 1:1000, Abcam), and GAPDH (ab8245, 1:5000, Abcam) before 1-h re-probing incubated with HRP-labeled goat anti-rabbit IgG (ab6721, 1:5000, Abcam) or goat anti-mouse IgG (ab6789, 1:5000, Abcam) at ambient temperature. The membrane was developed with luminescent liquid. The protein was qualified using ImageJ software (National Institutes of Health) as normalized to internal reference (GAPDH).

Cells were lyzed in RIPA buffer supplemented with proteasome and phosphatase inhibitors (Beyotime). Equal amounts of proteins were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight at 4°C with antibodies specific to GAPDH (1:1,500; CST#5174, Cell Signaling Technology, Danvers, MA, United States), COL1A1 (1:1,000; ab34710, Abcam, Cambridge, United Kingdom), RUNX2 (1:1,600; ab192256, Abcam), active β-catenin (1:1,000; ab246504, Abcam) or total β-catenin (1:1,000; ab223075, Abcam). A stripping method was used to measure the two antibodies of same molecular weight. After washing four times (5 min each time) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (Beyotime) for 1 h at room temperature. After washing three times (5 min each time) with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, United).

Bone marrow-derived mesenchymal stem cells (BM-MSCs) were isolated and cultured as previously described [23 (link)]. The human OS cell lines MG63, Saos2, HOS, and U2OS were purchased from the American Type Culture Collection (ATCC). BM-MSCs and human OS cell lines were cultured in DMEM (CR-12800, Cienry) supplemented with 10% FBS (11011-8611, Hangzhou Sijiqing Co., Ltd), penicillin (50 U/mL; Gibco), and streptomycin (50 µg/mL; Gibco), and cells were maintained in humidified 37 °C incubators with 5% CO₂. Antibodies against CCR9 (ab32556), total β-catenin (ab68183), active β-catenin (ab246504), and GAPDH (ab9485) were purchased from Abcam. Antibodies against E-cadherin (sc-8426), N-cadherin (sc-59987), vimentin (sc-6260), Twist (sc-81417), Snail (sc-271977), and MMP-1 (sc-21731) were obtained from Santa Cruz Biotechnology. Primary antibodies were used for western blotting at a dilution of 1:1000, and secondary antibodies were used at a dilution of 1:3000. The primary antibodies and the secondary antibodies for immunofluorescence were used at dilutions of 1:400 and 1:1000. The pathway inhibitor XAV-939 (HY-15147, Med Chem Express, Inc.) was used to inhibit the Wnt/β-catenin signaling pathway as previously described [24 (link)].