To obtain cell lysates, cells were washed twice with ice-cold PBS and disrupted in RIPA lysis buffer (50 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing 2 mM sodium orthovanadate, phosphatase inhibitor cocktail (Merck, Darmstadt, Germany), and protease inhibitor cocktail (Roche, Basel, Switzerland). The cell lysates were centrifuged for 30 minutes at 16,100g at 4°C and the supernatant was collected. Proteins in cell lysates were resolved by SDS-PAGE and then transferred to PVDF membranes. Nonspecific binding sites were blocked according to the manufacturers' instructions or with nonfat-dried milk (Nestle, Switzerland), if not indicated by the manufacturers specifically. The membranes were probed with primary antibodies overnight at 4°C. The following primary antibodies were used to probe the bound proteins: anti-Erk (1:4000); anti-phosphorylated-Erk (1:4000); anti-p38 (1:1000); anti-phosphorylated-p38 (1:1000); anti-JNK (1:1000); anti-phosphorylated-JNK (1:2000); anti-Akt (1:1000); and anti-phosphorylated-Akt (1:1000; Cell Signaling, Danvers, MA) and anti-p53 (1:1000); and anti-GAPDH (1:10,000) antibodies (Santa Cruz, CA). Secondary HRP-conjugated antibodies (1:2000; Vector Laboratories, Burlingame, CA) specific to the primary antibodies were applied followed by an extensive washing procedure again. The targeted proteins were visualized with enhanced chemiluminescence detection kits (Amersham, UK). Semi-quantitative measurements of the Western blotting results were performed using lab analysis software (TotalLab TL120; TotalLab, Tyne and Wear NE1, UK). The expression levels of the proteins were normalized and values of the controls were denoted as 100% with respect to hRPE1 or hRPE3 cells.