Ab109420

Proteins were separated by SDS-PAGE and transferred to PVDF membrane for Western blotting. Membranes were blocked in 5% w/w non-fat milk powder in PBS-T. Primary antibodies used for blotting were: MEF2A (rabbit monoclonal, 1:5000, Abcam ab109420), GluA1 (rabbit polyclonal, 1:1000, Millipore AB1504), GluA2 (rabbit polyclonal, 1:2000, Synaptic Systems 182103), GAPDH (mouse monoclonal [6C5], 1:10000, Abcam ab8245), Arc (rabbit polyclonal, 1:1000, Synaptic Systems 156003), mGluR1α (rabbit polyclonal, 1:500, Millipore AB1551), mGluR5 (rabbit polyclonal, 1:5000, Upstate Biotechnology 06–451). For protein detection, membranes were incubated with HRP-conjugated secondary antibodies and visualised by enhanced chemiluminescence. Protein bands were quantified by densitometry using ImageJ (NIH). For total protein expression, band intensity of the protein of interest was normalised to the loading control. For proportional surface expression, surface band intensity of the protein of interest was normalised to its band intensity in the total lysate.

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.