Ab265600

The procedures of Western blot were adopted from a previous study [33 (link)]. RIPA lysate was utilized for extracting total proteins from cells. Cells suspended in RIPA buffer were lyzed on ice for 10 mins and lysed cells were centrifuged at 14,000 rpm for 10 mins. The supernatant containing total protein lysate was quantified by a BCA Protein assay kit (Beyotime Biotechnology P0009; Shanghai, China). Aliquots of proteins (30 µg) were seperated through 10% SDS-PAGE, followed by the transfer of proteins onto PVDF membranes. After blocking with 5% skimmed milk for 1 hour, the membrane was then incubated with primary antibodies overnight at 4°C.: B-cell lymphoma-2 (Bcl-2) (1:500; ab196495, Abcam, Cambridge, UK), BCL2-associated x protein (Bax) (1:1000; ab32053, Abcam), Cleaved caspase 3 (1:1000; ab231289, Abcam), MAPK1 (1:800; ab265600, Abcam) and β-actin (1:5000; ab179467, Abcam). The membrane was washed 3 times with TBST for 5 minutes each. After wash, the membrane was further incubated with goat anti-rabbit IgG H&L (Abcam; ab96899, 1:5000) for additional 1 h under ambient temperature. Enhanced chemiluminescence ECL reagent (Pierce, IL, USA) was employed to visualize protein bands. The protein bands image was captured using Gel Doc 100 system and the Quantity One® imaging software (Bio-Rad, CA, USA).

Cells were collected by cell scraping. RIPA lysis buffer rich in protease inhibitors (Beyotime, China) was applied to extracted the proteins. BCA protein quantification kit (Beyotime, China) was applied to detect the proteins concentrations following the instructions of the manufacturer. Proteins were separated by electrophoresis using SDS-PAGE (Beijing Biosynthetic Biotechnology, China) and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, United States). The PVDF membrane was put into 5% skimmed milk powder blocking solution (Solarbio, China), and sealed for 2 h at room temperature. The sealed PVDF membrane was incubated with the anti-OCN (1:1000; Abcam, ab133612), OPN (1:1000; Abcam, ab214050), RUNX2 (1:2000; Abcam, ab76956), Collagen X (1:1000; Abcam, ab182563), MAPK1 (1:5000; Abcam, ab265600), GAPDH (1:2500; Abcam, ab9485), MEF2A (1:1000; Abcam, ab109420) overnight at 4°C.According to the source of the primary antibody, the secondary antibody was diluted with TBST solution and the PVDF membrane was put into the corresponding secondary antibody. An ultra-sensitive ECL chemiluminescence kit was applied to expose the membrane. The chemiluminescence of the protein bands was quantified by ImageJ software.