Biomax film

Synaptosomes were prepared from adult mice (>P60, n = 3 per group) as previously described [18 (link)]. In some experiments, synaptosomes (10 μg protein) were diluted in RIPA buffer followed by the addition of SDS gel sample buffer and analyzed as follows. Briefly, 10 μg of hippocampal synaptosomal protein was applied to SDS-PAGE gels followed by transfer to Immobilon P membranes (Millipore, Billerica, MA). After blocking membranes with 1× PBS, 0.1% Tween-20, and 5% milk, proteins were visualized by primary antibody [1:4000 NRP2 antiserum, gift of Alex Kolodkin, 1:1000 NeuN from AbCam, 1:1000 semaphorin 3F from AbCam], followed by secondary antibody detection with horseradish peroxidase (HRP)-linked anti-rabbit or anti-mouse IgG (1:5000; Jackson ImmunoResearch) using Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). Light emission was detected by BioMax film (Sigma-Aldrich, St Louis, MO). Optic densities (OD) of detected bands were expressed as a mean of the ratios of experimental versus wild-type mouse values ± SEM. Statistical analysis was performed using ANOVA with post hoc Tukey’s multiple comparison test analysis.

Whole protein extracts from human fibroblast cells were resuspended with lysis buffer and protein concentration was estimated by the Bradford Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA). Proteins, 60 mg per lane, were separated by SDS-PAGE, transferred to nitrocellulose membranes (Amersham) and incubated with the REEP4 (Abcam, Cambridge, UK) primary antibody. After removal of the unbound primary antibody, the membrane was incubated with secondary antibody-peroxidase conjugate (Sigma-Aldrich, Saint Louis, MO, USA), processed for detection by chemiluminescence (Amersham, GE Healthcare, Lafayette, CO, USA) and imaged on Biomax film (Sigma-Aldrich). Actin antibody was used as internal control.