Rabbit pab to γ2

Density gradient ultracentrifugation was performed as described elsewhere23 (link). Mouse cortical neurons were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer D (25 mM HEPES–KOH (pH 6.8), 150 mM NaCl, 2 mM EDTA, 1% digitonin, protease inhibitor cocktail and phosphatase inhibitor cocktail) at 4 °C for 60 min. After centrifugation at 17,000g for 30 min, the supernatant (1.5 mg of protein per 0.5 ml) was layered over an 11.5-ml 10–40% (w/v) linear sucrose density gradient containing 25 mM HEPES–KOH (pH 6.8), 150 mM NaCl and 0.4% digitonin. After centrifugation for 15 h at 35,000 r.p.m. in a Beckman SW40 rotor, 12 fractions each containing 1 ml were collected from the top of the tube and subjected to immunoblotting or immunoprecipitation. The antibodies used in immunoblotting were as follows: rabbit pAb to α1 (1:1,000; Synaptic Systems); rabbit pAb to γ2 (1:1,000; Synaptic Systems); rabbit pAb to 14-3-3ζ (1:100; Santa Cruz Biotechnology); mouse mAb to 14-3-3θ (1:5,000; SIGMA); mouse mAb to dynactin1 (1:250; Transduction Laboratories); and mouse mAb to KIF5 (1:200; Millipore). Protein mobility markers (high-molecular-weight native marker kit; GE Healthcare) were applied to a parallel gradient, and their fraction positions were determined by 2–15% native PAGE, followed by Coomassie brilliant blue staining.

Biotinylation of neuronal surface proteins was performed as described previously23 (link). PX-RICS^+/+ and PX-RICS^−/− cortical or hippocampal neurons (14 DIV) or CGNs (10 DIV) were washed three times with ice-cold PBS and incubated with 0.25 mg ml⁻¹ EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific) in PBS for 20 min at 4 °C with gentle agitation. After an immediate rinse with ice-cold quenching buffer (50 mM glycine in PBS), the neurons were further washed three times in ice-cold quenching buffer for 5 min each. The cells were lysed in lysis buffer T (10 mM Tris-HCl (pH 6.8), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail and phosphatase inhibitor cocktail) and cleared lysates (0.5 mg of protein) were incubated with 60 μl of 50% slurry of streptavidin–agarose beads (Thermo Scientific) for 2 h at 4 °C. Beads were washed five times in lysis buffer T and then once with PBS. Bound proteins were analysed by immunoblotting with rabbit pAb to γ2 (1:1,000; Synaptic Systems), mouse mAb to transferrin receptor (1:500, Zymed) and mouse mAb to α-tubulin (1:500; Calbiochem). Band intensities were quantified using ImageJ software.