Enhanced chemiluminescence western blotting reagents

p53 protein expression was assessed by western blotting. The cells were lysed in radioimmunoprecipitation assay buffer containing 150 mM NaCl, 50 mM Tris-Cl (pH 7.6), 5 mM EDTA, 0.5% NP-40, 0.5% Triton X-100 containing 10 µg/ml each of leupeptin, aprotinin, and antipain, 1 mM sodium orthovanadate and 0.5 mM phenylmethanesulfonyl fluoride (all Beyotime Institute of Biotechnology, Shanghai, China). The protein concentration was determined using the bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). Total protein (50 µg) was loaded onto each lane, separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). After being blocked with 5% skimmed milk in Tris-buffered saline (pH 7.6; Beyotime Institute of Biotechnology) at room temperature, the membrane was incubated at 4°C overnight with rabbit anti-p53 (1:1,000) and anti-β-actin (1:1,000) primary antibodies. Following incubation with HRP-conjugated goat anti-rabbit secondary antibody (1:5,000), the immune complexes were detected by enhanced chemiluminescence western blotting reagents (Beyotime Institute of Biotechnology). Data were normalized to β-actin protein expression levels and relative protein expression levels were determined using AlphaView Stand Alone Analysis Software 3.0 (ProteinSimple, San Jose, CA, USA).

ADSCs of the co-culture system which were seeded on the bottom were used for the experiments. Proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (P0028, Beyotime Institute of Biotechnology, Haimen, China) and detected by BCA Protein Assay Kit (P0009, Beyotime Institute of Biotechnology). Protein extracts (50 µg per lane) were resolved using 10% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes. Then PVDF membranes were incubated with PBS containing 5% nonfat milk and 0.1% Tween 20 at room temperature for 1 h, and blocked with 2.5% normal bovine serum at room temperature for 1 h. Following this, the membranes were probed with primary antibodies againstanti-S-100 (rabbit monoclonal; 1:1,000; cat. no. ab52642; Abcam), GFAP (rabbit monoclonal; 1:1,000; cat. no. ab68428; Abcam) and GAPDH (rabbit polyclonal; 1:2,500; cat. no. ab9485; Abcam) at 4°C overnight. The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. A0208; 1:5,000; Beyotime Institute of Biotechnology) at room temperature for 1 h, following three washes with TBS and 0.1% Tween-20. The immuno blots were detected using Enhanced Chemiluminescence western blotting reagents (Beyotime Institute of Biotechnology) after washing, and the image was scanned with a GS800 Densitometer Scanner.