En rage

Cells (1 × 10⁵) were seeded in 10-cm plates and maintained in 5% FBS-MEM until 70–75% confluent. The medium was changed to serum-free medium 24 h prior to the treatment of cells with the RAGE neutralizing antibody (10 μg/ml, R & D Systems, Minneapolis, MN, Cat#AF1145) or EN-RAGE (10 μg/ml, R & D Systems, Cat#1052-ER) for 4 h. After this treatment, the cells were seeded on AGE-modified or unmodified BME. To confirm the binding of EN-RAGE, treated cells were washed three times with 1X PBS and the cell lysate (prepared using RIPA buffer) was analyzed by Western blotting for EN-RAGE.

Whole cell lysates were prepared using the Mammalian Protein Extraction Reagent (Thermo scientific, Rockford, IL) containing a 1:100 diluted protease and phosphatase inhibitor cocktail (Sigma-Aldrich) (for αSMA, Fibronectin, phosphorylated and total Smad2 and ERK1/2) or with RIPA buffer (Cell Signaling Technologies, Danvers, MA) containing 1 mM PMSF (Sigma-Aldrich) (for RAGE and EN-RAGE detection). Proteins (10 to 20 μg) were separated on 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated at 4 °C overnight with primary antibodies against the following proteins: RAGE (1 μg/ml, R & D Systems), EN-RAGE (1 μg/ml, R & D Systems, Cat#AF1052), αSMA (1:5000 dilution, Sigma-Aldrich, Cat#A5228), Fibronectin (1:200 dilution, Santa Cruz Biotechnology, Dallas, TX, Cat#sc9068), phosphorylated (phospho S245/S250/S255, 1:1000 dilution, Cell Signaling, Cat#3104) Smad2, total Smad2 (1:1000 dilution, Cell Signaling, Cat#5339), phosphorylated (phospho T202/Y204, 1:1000 dilution, Cell Signaling, Cat#4370) ERK1/2, total ERK1/2 (1:1000 dilution, Cell Signaling, Cat#9212) and β-Actin (1:1000 dilution, Cell Signaling, Cat#4970). Appropriate HRP-conjugated secondary antibodies (1:5000 dilution, Cell Signaling) were used. The protein bands were detected using the SuperSignal West Pico or Femto Kit (Pierce Chemicals, IL).