Chemiluminescence kit

A western blot assay was performed as has been previously described^{32 (link)}. In brief, whole-cell extracts were sonicated in lysis buffer and homogenized. Samples containing 30–50 µg total protein were resolved on 8–12% polyacrylamide–SDS gels and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk, incubated with primary antibody, then incubated with an alkaline phosphatase-conjugated secondary antibody. Protein bands were detected with a chemiluminescence kit (Roche Diagnostics Corp., Indianapolis, IN, USA).

Proteins (20 μg) were separated in 12% sodium dodecyl sulfate-polyacrylamide gels. After blocking with 5% non-fat milk for 1 hr, membranes so obtained were incubated with primary antibodies (all diluted 1:1000) for anti-IκB-α (Santa Cruz Biotechnology, Santa Cruz, CA), anti-α-tubulin (Cell Signaling, Danvers, MA), anti-p65 (Santa Cruz Biotechnology), anti-Lamin B (Santa Cruz Biotechnology), anti-P-selectin (Santa Cruz Biotechnology), anti-ICAM-1 (Santa Cruz Biotechnology), anti-PSGL-1 (Santa Cruz Biotechnology), and anti-CD11b (BD Transduction Laboratories, San Jose, CA) overnight at 4 °C. The next day, blots were detected using a chemiluminescence kit (Roche, Basel, Switzerland) using HRP-conjugated secondary antibodies (1:2000; Santa Cruz Biotechnology).

Blood was obtained from all participants after fasting for 8 h. The CA72-4 level was assessed using a chemiluminescence kit (Roche Diagnostics GmbH). Serum CEA, CA19-9, and AFP levels were respectively measured using chemiluminescence assays (Beckman Coulter, Fullerton, CA, USA). The cutoff values for serum CEA, CA19-9, AFP, and CA72-4 were 5 ng/mL, 37 U/mL, 20 ng/mL, and 6.9 ng/mL, respectively, and were measured according to the manufacturer’s instructions.

Genomic DNA was isolated by lysing 50 mg of tissue with lysis buffer (100 mM TRIS-HCL pH 8.5; 0.2% SDS; 5 mM EDTA; 200 mM NaCl) and Proteinase K (10 mg/mL) and incubated overnight at 55–60 °C in a shaker. After centrifugation (15 min at 21,000× g) 560 µL of saturated NaCl (5.5 M) was added to 400 µL of the supernatant followed by another centrifugation step (15 min with 14,000 RPM) for protein precipitation. 700 µL of 100% ethanol was added to the supernatant and mixed thoroughly for DNA precipitation. The pellet was washed several times in 70% ethanol, dried at room temperature and resuspended in 50 µL H₂O at 37 °C overnight. DNA was diluted if the concentration was above 2 µg/µL. After digestion with NcoI, the DNA was then electrophoresed, blotted, and hybridized to a DIG-labeled probe against EGFP. After washing, the probe was visualized using a chemiluminescence kit (Roche Diagnostics, Mannheim, Germany) and a Vilber Lourmat Fusion-SL3500 documentation system. Flanking DNA fragments were expected to be >3 kb.

Protein extracts were obtained from articular cartilage, osteophyte cartilage, and bone as described previously [19 (link)]. After boiling, 50 μg protein extracts were separated by sodium dodecyl sulfate (SDS) PAGE on a 10% gel and transferred on nitrocellulose transfer membranes. The efficiency of protein transfer was controlled by Ponceau S staining. After blocking, the nitrocellulose membranes were incubated with a monoclonal mouse anti-PEDF antibody diluted 1 : 1250 (Abcam, Cambridge, UK) or a rat anti-α-tubulin antibody (diluted 1 : 1000) (AbD Serotec, Kidlington, UK) overnight at 4°C. Horseradish peroxidase-conjugated goat anti-mouse antibodies (GE Healthcare, Little Chalfont, UK) or goat anti-rat antibodies (Dianova, Hamburg, Germany) (diluted 1 : 10000) were used as secondary antibodies. The immunoreactive proteins were visualized using a chemiluminescence kit (Roche, Mannheim, Germany) followed by exposure to a chemiluminescent detection film.