Ponceau s staining solution

Cells were lysed by RIPA Lysis Buffer (Beyotime Biotechnology, China). The proteins are electrophoretic separated with 11% SDS‐PAGE gel, transferred to PVDF membrane (Millipore, USA) and stained with Ponceau S staining solution (Beyotime Biotechnology, China) for 5‐10 minutes. After blocking with 5% evaporated skimmed milk, the membranes were incubated with each primary antibody, including anti‐TSG101, anti‐Calnexin, anti‐RUNX2, anti‐Osterix, anti‐Wnt1, anti‐β‐catenin, anti‐Axin2 (Abcam, 1:1000 dilution) for 16 hours and the respective secondary antibody (Cell Signaling Technology, 1:5000 dilution). After the membranes were washed with TBST three times, the target bands were detected by ECL kit (Solarbio, China). The relative intensity was measured by normalization using GAPDH.

Proteins were separated by electrophoresis on 11% SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA) and stained with Ponceau S staining solution (Beyotime Biotechnology, China) for 5-10 min. Membranes were then incubated with each primary antibody, including anti-ALP, anti-RUNX2, anti-TSG101, anti-CD9, and anti-calnexin (Abcam, USA, 1 : 1000 dilution) for 16 h, and the respective secondary antibody (Cell Signaling Technology, USA, 1 : 5000 dilution) after the membrane was blocked with 5% evaporated skimmed milk. After each incubation, the membrane was washed three times with TBST. Target bands were developed using an enhanced chemiluminescence (ECL) kit (Solarbio, China) according to the manufacturer's instructions. To quantify the results of western blots, we calculated mean Intden (integrated density) values using ImageJ 1.8.0 software. The relative intensity was measured by normalization using GAPDH.

Protein samples were subjected to immunoblot analysis. After the electrophoresis and transmembrane, polyvinylidene fluoride membrane was stained by Ponceau S staining solution (Beyotime) for 30 min. The membrane was washed and used for scanning.