Western blot analysis of inflammatory factors in whole cell lysate

Cell proteins of RAW 264.7 cells were collected. Briefly, cells were plated at a density of 2×10⁵ cells/dish in a 100 mm culture dish and incubated with 10 µM ZA for 24 h, followed by incubation with 0.1 µg/ml LPS and various concentrations of PDRN (1, 10 or 100 µg/ml) in serum free medium at 37°C for 24 h. Cells were harvested and gently homogenized in radioimmunoprecipitation assay buffer consisting of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂ EDTA, 1 mM EGTA, 1% Nonidet P-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin (Cell Signaling Technology, Inc., Danvers, MA, USA) and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich; Merck KGaA). Subsequently, the lysates were incubated for 20 min at 4°C, centrifuged at 16,000 × g for 20 min at 4°C, following which they were rapidly frozen. The lysates were measured for protein quantification using Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 30 µg/lane protein was subjected to western blot analysis using 12% SDS-PAGE gel and transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Little Chalfont, UK). Membranes were incubated in a blocking buffer containing 5% skim milk for 1 h at room temperature. They were subsequently incubated overnight at 4°C with the following primary antibodies: Mouse β-actin, inducible NO synthase (iNOS), TNF-α and VEGF (cat. nos. SC-4778, SC-7271, SC-52746 and SC-7269, respectively), rabbit A_2A (cat. no. 13937; all 1:1,000; Santa Cruz Biotechnology Inc., Dallas, TX, USA), IL-1β and IL-6 (cat. nos. bs-6319r and bs-0782r, respectively; both 1:1,000; Bioss Antibodies, Inc., Woburn, MA, USA). The membranes were then incubated at room temperature for 1 h with anti-mouse and anti-rabbit secondary horseradish peroxidase-conjugated antibodies (cat. no. PI-2000 and PI-1000, respectively; both 1:1,000; Vector Laboratories, Inc., Burlingame, CA, USA). Bands were detected using an enhanced chemiluminescence detection kit (Bio-Rad Laboratories, Inc.). Densitrometric analysis of the resultant bands was performed using Molecular Analyst^™ software, version 1.4.1 (Bio-Rad Laboratories, Inc.).