Enhanced chemiluminescence assay kit

The cells were treated as above and lysed in lysis buffer. Protein concentrations were determined using a commercial kit (Bradford Protein Assay, Biorad, UK) according to the manufacturer's specifications. Proteins were separated on 12% polyacrylamide gels and transferred to polyvinylidenefluoride membranes. These membranes were incubated for 1 h at room temperature in Tris-buffered saline containing 0.5% non-fat milk powder, and 0.1 % Tween-20. An antibody against vimentin (Abcam, Cambridge, UK), an antibody against p53 (Abcam, Cambridge, UK), and an antibody against bcl-2 (Abcam, Cambridge, UK) were used. An antibody against GAPDH (diluted 1:1,000; Abcam, Cambridge, UK) was used as the positive control. After incubation with the primary antibody overnight at 4 °C, the membranes were washed three times in TBS. After incubation with the secondary horseradish peroxidase-conjugated antibodies (1:1,000; Abcam, Cambridge, UK) for 1 h at room temperature, detection was performed using an enhanced chemiluminescence assay kit (GE Healthcare, UK). Digital images were captured and density measurements were made using commercial software (Quanity One, Biorad).

BMSCs were treated with various concentrations of naringin (0.1, 1, and 10 µM) for 48 h. The protein expression levels of PPARγ were then determined by western blotting. The protein concentrations were determined using a Novagen ® Bicinchoninic Acid Protein Assay kit (Novagen, Merck Millipore, Darmstadt, Germany). Protein was extracted from cells using Cell Lysis Buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). Equal quantities of protein was separated by 10% SDS-polyacrylamide gel and blotted onto polyvinylidene fluoride membranes (Millipore). The primary antibodies used for the western blot analysis were as follows: Anti-PPARγ (cat. no. 2435S; 1:500; Cell Signaling Technology, Inc.) and anti-β-actin (cat. no. sc-69879; 1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4˚C overnight. Subsequently, membranes were washed and incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (cat. no. C520011; 1:20,000, Sangon Biotech Co., Ltd., Shanghai, China). The blots were visualized using an enhanced chemiluminescence assay kit (GE Healthcare Life Sciences, Little Chalfont, UK), and analyzed using VersaDoc Gel Imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The cells were lysed within the wells using lysis buffer (60 mM Tris, 2 % SDS, 100 mM DTT) on ice for 10 min and protein concentrations were then determined with a commercial kit (Bradford Protein Assay, Biorad, UK). Samples were subjected in 10 % SDS polyacrylamide gel and separated proteins were transferred to a PVDF membrane. The blots were blocked with blocking buffer (Tris-buffered saline, 0.5 % skim milk powder, and 0.1 % Tween-20) for 1 h at room temperature, and subsequently incubated overnight at 4°C with primary antibody against catalase with anti-catalase (1:2000; Abcam, Cambridge, UK), and mouse monoclonal anti-GAPDH (1:1,000; Abcam, Cambridge, UK). The blots were washed three times in TBST (TBS, 0.1 % Tween-20). The blots were then incubated with the secondary horseradish peroxidase-conjugated antibodies (1:1,000; Cell Signaling Technology, UK) for 1h at room temperature. The bands were visualized using enhanced chemiluminescence assay kit (GE Healthcare, UK) in the Biorad ChemiDoc MP imaging system and density measurements were made using commercial software (Bio-Rad Quantity One).