Ab205394

Tissues and cells are pyrolyzed in a protein lysis system containing RIPA, PMSF (Wuhan Boster Biological Technology, Ltd, Wuhan, China), and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Protein concentration was measured at 562 nm. A total of 30 μg of proteins were presented in SDS-PAGE gel system then separated and transferred to polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Bedford, MA, USA) for 90 min. After transfer to the PVDF membrane, the membrane was blocked in PBS containing 5% skim milk for 1 h and then incubated with antibodies against BMP1 (1:1000; ab205394; Abcam Co., Ltd) and GAPDH (1:2000; BM3876; Wuhan Boster Biological Technology, Ltd, Wuhan, China) at 4 °C overnight. The next day the membranes were washed and incubated with secondary antibodies (1:5000; BA1020; Wuhan Boster Biological Technology, Ltd, Wuhan, China) at room temperature (25 °C) for 2 h. Finally, the membranes were washed and detected by Biosense SC8108 Gel Documentation System with GeneScope V1.73 software (Shanghai BioTech, Shanghai, China).

The proteins were isolated using radioimmunoprecipitation assay lysis buffer (Thermo Fisher) and quantified using the BCA Kit (BioVision, USA). After separation by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and loading onto polyvinylidene fluoride membranes (Thermo Fisher), the proteins were blocked with 5% milk and incubated with primary antibodies overnight and a secondary antibody conjugated with horseradish peroxidase (BD Biosciences) at room temperature for 1 h. Protein banding was evaluated using the SynGene system and GeneSnap software (SynGene, USA). Primary antibodies: Glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), Periostin (ab14041, Abcam), BMP1 (ab205394, Abcam), osteopontin (OPN) (AB5405, MilliporeSigma), runt-related transcription factor 2 (RUNX2) (ab23981, Abcam).

After dewaxing and dehydration, the sections were immersed in 3% H₂O₂ to inactivate the endogenous enzymes. After treatment with 5% BSA for 20 min, the sections were added with Periostin (ab14041, Abcam) and BMP1 (ab205394, Abcam) at 4 °C overnight, horseradish peroxidase-labeled secondary antibody at 37℃ for 30 min, and streptavidin–biotin complex at 37℃ for 20 min. Following color development using diaminobenzidine, the sections were stained with hematoxylin, dehydrated and permeabilized with xylene, and sealed with neutral balsam.