Image pro plus 5

Cells were plated at a density of 3 × 10⁵ cells per 100 mm culture dish in 10 mL of DMEM/F-12 containing 5% FBS. At 24 h after 3 HBO interventions, the cells were washed with PBS and extracted using M-PER Protein Extraction Reagent (Thermo, USA). The protein content was quantitated using a protein assay kit (Pierce Biotechnology, Rockford, IL, USA), separated by 7.5% SDS-PAGE, and transferred onto membranes using a transfer unit (Bio-Rad, USA). After blocking, the membranes were incubated with 1000-fold diluted rabbit antibodies against Wnt3a, phosphor-LRP6 (Cell Signaling), LRP6 (Abcam, Cambridge, UK), cyclin D1/2 (Merck, Darmstadt, Germany), mouse antibodies against β-catenin (Millipore, Temecula, CA, USA), and β-actin (Millipore). After washing, the membranes were further incubated for 2 h with 10,000-fold goat anti-mouse IgG (Calbiochem, USA) or goat anti-rabbit IgG (Millipore) conjugated to horseradish peroxidase. The membranes were then washed and rinsed with ECL detection reagents (Millipore). The bands were photographed using ECL Hyperfilm (Amersham Pharmacia UK) and their intensity was quantified using an image-analysis system (Image-Pro Plus 5.0).

After culturing for 7 or 14 days, the cells were washed with PBS and extracted using M-PER protein extraction reagent (Thermo, USA). The protein content was quantitated using a protein assay kit (Pierce Biotechnology, IL), separated by 7.5 % SDS-PAGE, and transferred onto membranes using a transfer unit (Bio-Rad, USA). After blocking, the membranes were incubated with 1000-fold diluted rabbit antibodies against leptin (R&D, MN, USA), leptin-R (R&D), Runx2 (Millipore, Temecula, CA), and β-actin (Millipore). After washing, the membranes were further incubated for 2 h with 10,000-fold goat anti-mouse IgG (Calbiochem, USA) or goat anti-rabbit IgG (Millipore) conjugated to horseradish peroxidase. The membranes were then washed and rinsed with ECL detection reagents (Millipore). The bands were photographed using ECL Hyperfilm (Amersham Pharmacia Biotech, UK), and their intensity was quantified using an image analysis system (Image-Pro plus 5.0).