P mapk

The corneal tissue was lysed in ice-cold RIPA buffer with protease inhibitors, and homogenized by ultrasound. Protein concentration was determined by BCA method. The same amounts of protein were subjected to SDS-PAGE electrophoresis, and transferred to PVDF. The membrane was blocked with 2% BSA for 1 h, and blotted with primary antibodies against p-VEGF2 (1:200; Santa Cruz, USA), t-VEGF2 (1:200; Santa Cruz, USA), STAT3 (1:200; Santa Cruz, USA), p-MAPK (1:200; Santa Cruz, USA), t-MAPK (1:200; Santa Cruz, USA), p-Akt (1:200; Santa Cruz, USA), t-Akt (1:200; Santa Cruz, USA), β-actin (1:10 000; Bio-Rad, Hercules, CA, USA), VEGF (1:200; Santa Cruz, USA) and PDGF (1:200; Santa Cruz, USA) overnight at 4 °C. After washing with TBST, horseradish peroxidase-labeled secondary antibody (1:5000; Dako, Shanghai, China) was added and incubated for 1 hour at room temperature. After washing with TBST and incubating with ECL reagent, film was used for signal development. ImageJ software was used to scan the gray value of each band, with β-actin as the reference control. Each experiment was repeated 3 times.