Horseradish peroxidase conjugated cholera toxin b subunit

Lipid raft fractionation was conducted following the previous method 13 (link) using OptiPrep (Sigma) after 293T cells were transfected with the EGFP-NEGR1. After lysates were adjusted to 40% OptiPrep, the samples was loaded into a centrifuge tube, which was serially overlaid with 2.5 ml of 28% OptiPrep in 1 × PBS and 0.6 ml of 1 × PBS. After centrifuging for 3 h at 36,400 rpm in a Beckman SW 55 Ti rotor, 13-300 μl fractions were collected from the top. A horseradish peroxidase-conjugated cholera toxin B subunit (Sigma) was used to detect Ganglioside GM1, a raft marker. The crude membrane was obtained according to a previously described method 14 (link). For enzyme digestion, samples were incubated with 500 U of PNGase F or EndoH (New England Biolabs, Ipswich, USA) at 37°C for 1 h. A neurite outgrowth assay was performed using undifferentiated SHSY-5Y human neuroblastoma cells. At 24 h after seeding of 5 × 10⁴ cells into 6-well cell culture plate, 100 ng/ml of either NEGR1-hFc or hFc were added to the culture media and incubated for five days. Cells were also treated with retinoic acid (10 µM) as a positive control. Microtubule-associated protein 2 (MAP2), a marker of neuronal differentiation, was also detected using anti-MAP2 antibody (Sigma-Aldrich).