Cebp α

Total proteins were extracted by RIPA buffer and measured using the BCA method from BMSC in each group. SDS-PAGE was performed on a 10% polyacrylamide gel and samples were transferred onto PVDF nitrocellulose membranes (Millipore, Bedford, MA) under semi-dry conditions. The membranes were blocked overnight with 5% non-fat milk, then incubated with primary antibodies at 4 °C overnight, including: CD9 (1:1000, #13403, CST, Boston, MA),CD63 (1:1000, sc-5275, Santa Cruz, Dallas, TX), RUNX2 (1:1000, sc-101145, Santa Cruz, Dallas, TX), Osterix (1:1000, ab944744, Abcam, Cambridge, UK), CEBP-α (1:1000, AF6333, Affinity, Cincinnati, OH), PPARγ2 (1:1200, sc-7273, Santa Cruz, Dallas, TX), SPRY2 (1:1000, #14954, CST, Boston, MA), p-JNK (1:1000, #9255, CST, Boston, MA), JNK (1:1000, #9252, CST, Boston, MA), p-p38 (1:1000, #4511, CST, Boston, MA), p38 (1:1000, #8690, CST, Boston, MA), ERK1/2 (1:800, #11257-1-AP, Proteintech, Rosemont, IL), p-ERK1/2 (1:2000, #4370, CST, Boston, MA), GAPDH (1:1500, #5174, CST, Boston, MA). Following this, membranes were incubated with secondary horseradish peroxidase-conjugated antibodies at room temperature for 2 h. Lastly, the proteins were analysed using the luminescent image analyser Image Quant LAS-4000 Mini (GE Healthcare, Chicago, IL). All reactions were repeated three times and data were analysed with Image J software (Bethesda, MD).