Enhanced chemiluminescent reagent

After lysed in the protease inhibitors contained RIPA buffer (Sigma-Aldrich), total proteins of OS cells were isolated by high-speed centrifuge at 12,000 g/min. BCA protein assay kit (Thermo Fisher) was applied to determine the concentration of extracted proteins, and then 50 μg protein samples were loaded into and isolated by 10% SDS-PAGE (80 V, 30 min and 120 V, 45 min). Next, the isolated proteins were transferred into 0.45-μm polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA; 100 V, 60 min) followed by the incubation of 5% skimmed milk for 2 h. The membranes were incubated overnight with primary antibodies that against c-FLIP (cellular FLICE inhibitory protein, 1:800, ab8421, Abcam, UK), E-cadherin (1:500, ab15148, Abcam, UK), N-cadherin (1:1000, ab76057, Abcam, UK), Vimentin (1:2000, ab137321, Abcam, UK), Snail (1:1000, ab180714, Abcam, UK) and GAPDH (1:10,000, ab181602, Abcam, UK). GAPDH was loaded as an internal reference. After removing the excessive primary antibodies, the membranes were subjected for incubation of horseradish peroxidase conjugated secondary antibodies (IgG-HRP, Abcam, 1:2000, UK) for 2 h, and bands were detected by an enhanced chemiluminescent reagent (ECL, Germany). And the relative protein expression level was quantified using the Image J software.

After two washes with PBS, the cells were treated with radioimmunoprecipitation buffer (Beyotime) containing 1 mmol L⁻¹ phenylmethyl sulfonyl fluoride. Following a 1 minute ultrasonic shock, the MC3T3-E1 cells were centrifuged at 12 000g and stored at 4 °C for 10 minutes. The cell fragments were then discarded to obtain the total protein. The proteins were loaded onto a 7.5% SDS-polyacrylamide gel for electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was incubated in 5% bovine serum albumin for 2 hours and then with primary antibodies, including ALP (1 : 1000, Abcam, ab154100), RUNX2 (1 : 1000, Abclonal, A11753), β-catenin (1 : 1000, Abcam, ab265591), phosphorylation of β-catenin (1 : 1000, Abcam, ab75777), and GAPDH (1 : 2500, Abcam, ab9485), which were incubated overnight at 4 °C. The membrane was visualized using an enhanced chemiluminescent reagent (Abcam), and GAPDH was used as an internal control. Secondary antibodies (1 : 5000, ab6721, Abcam) were applied for 2 hours in the analysis.