Biotinylation assay

Biotinylation experiments were performed as previously described (Shimell et al., 2019). Briefly, neurons in 10 cm dishes were nucleofected with indicated constructs and experiments were carried out at 13 DIV. Neurons were washed with ice cold PBS-CM (0.1 mM CaCl₂ and 1 mM MgCl₂ in 1× PBS, pH 8) and incubated for 30 min with 0.5 mg/ml NHS-SS-Biotin in ice cod PBS-CM at 4°C with gentle rocking. After incubation, cells were washed once with PBS-CM and the unbound biotin quenched via two 8 min incubations with quenching buffer (20 mM glycine in PBS-CM). Lysis was performed using mechanical scraping in lysis buffer (1% IGEPAL-CA630 and 1mM PMSF with Roche Complete protease inhibitor tablet) and subsequently spun down at 500 g for 5 min at 4°C. Samples were vortexed, run through a 26 1/2-gauge syringe three times, and nutated at 4°C for 30 min. After nutation, samples were spun down at 16,100 g for 30 min at 4°C to clear the lysate. The cell lysate was then quantified for protein using a BCA assay kit (Thermo Fisher Scientific) as per the manufacturer's instructions. 10 mg of each whole-cell lysate was then combined with SDS-sample buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, 14.5 mM EDTA and 0.02% bromophenol blue with 1% β-mercaptoethanol), boiled for 5 min at 95°C and stored at −20°C as the input sample. 100–200 mg of the remaining protein sample was added to a 50 ml 50% slurry of Neutravidin-conjugated agarose beads (Thermo Fisher Scientific) that was pre-washed three times in lysis buffer. Each sample was then brought to a total volume of 500 ml with lysis buffer and nutated at 4°C overnight. The following day beads were pelleted and washed seven times using centrifugation (500 g for 3 min). Elution of the beads was performed using 40 ml of SDS-sample buffer with 100mM DTT. Samples were boiled at 90°C for 5 min and then run on a western blot with the whole-cell lysates.