Thy 1

RGC-5 lysates of were prepared with a lysis buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 1 mM EDTA, 10% glycerol, and 0.5% NP-40 and protease inhibitor cocktail (Invitrogen, USA). The total protein were then separated on 10% SDS-PAGE gel and transferred to the nitrocellulose membranes. We used primary antibodies against, Thy-1 (1:2000, Santa Cruz Biotechnology, USA), ERK1/2 (1:1000, Cell Signaling Technology, USA), phospho-ERK1/2 (pERK1/2, 1:2000, Cell Signaling Technology, USA), PI3k3 /serine-threonine kinase (AKT, 1:1000, Cell Signaling Technology, USA), phosphor-AKT (pAKT, 1:1000, Cell Signaling Technology, USA), TrkB (1:1000, Cell Signaling Technology, USA), phosphorylated TrkB (1:200, Cell Signaling Technology, USA) and IGF1R (1:200, Santa Cruz Biotechnology, USA). Horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, USA) were used and Actin was used as internal control. The western blots were visualized with an enhanced chemiluminesence system (Amersham Biosciences, USA) according to the manufacturer’s protocol. Band intensities normalized to the total protein immuno-precipitation under each control condition and quantified with an ImageJ software (NIH, USA).