Protein lysates were prepared by gently pipetting the retinal tissues up and down in a 50-mM (hydroxymethyl)aminomehane(Tris)-HCl (pH 7.5), 5 mM EDTA, 300 mM sodium chloride (NaCl), 0.1% nonyl phenoxypolyethoxylethanol (NP-40), and 1X protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA) several times and incubating for an hour in ice. Protein concentrations were quantified by the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 20 μg for each of the WT and NCAM
−/− proteins were adjusted with an equivalent volume of distilled water and mixed with 4x SDS-sample buffer (0.08% SDS, 250 mM Tris-HCl [pH 8.0], 40% glycerol, 20% β-mecaptoethanol, and a trace amount of bromophenol blue), boiled for 5 minutes, loaded in a polyacrylamide gel, ran for 2.5 hours at 80 V, then transferred to a polyvinylidene fluoride (PVDF) membrane at 0.25 A for 1.5 hours. Anti-PSA-NCAM (MAB5324, 1:2000; Millipore, Billerica, MA, USA), anti-NCAM (MAB310, 1:500; Millipore), anti-p75
NTR (8238, 1:1000; Cell Signaling Techology, Inc., Danvers, MA, USA),anti-Neurotensin Receptor 3 (Sortilin; 612100, 1:500; BD Transduction Laboratories, San Jose, CA, USA), anti-pro-NGF (AB9040, 1:2000; Millipore), and anti-actin (A2066, 1:5000; Sigma-Aldrich Corp., St. Louis, MO, USA) antibodies were used to determine protein expressions by standard SDS-polyacrylamide gel electrophoresis (PAGE) immunoblotting.
5 To capture active caspase 3 signals, protein lysates were loaded in a 15% polyacrylamide gel, separated via electrophoresis at 55 V for 5 hours and then transferred onto a PVDF membrane for 1 hour at 50 V. Anti-caspase 3 (cleaved) (AB3623, 1:100; Millipore) and anti-actin antibodies were used to determine protein levels. The chemiluminescent signals were detected by the Pierce ECL 2 Western Blotting Substrate (80196, Thermo Scientific, Rockford, IL, USA) and captured by exposure to X-ray films or by scanning with the Typhoon Variable Mode Imager (Amersham Biosciences, Sunnyvale, CA, USA). The levels of proteins were then quantified using Image J (
http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and the data obtained were converted to percentages of the controls.