Immunoprecipitation of p150Glued Protein

Mouse brains or ER microsomes fraction of mouse brains were homogenized in IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 50 mM NaF,10 mM glycerolphosphate, 2 mM EGTA, 2 mM EDTA, 1% NP-40, and 1× Protease and Phosphatase Inhibitor Cocktails) with Dounce homogenizer (10 strokes)^{54 (link)}. Lysates were centrifuged at 15,000 × g for 15 min at 4 °C, and the supernatants were collected. The protein concentration of the lysates was measured and adjusted to 1 mg/ml. After pre-clearing with Protein G agarose (Thermo Fisher Scientific), the lysates were incubated with antibody-bound Protein G agarose for 1 h at 4 °C. After five washes of the agarose beads with IP buffer at 4 °C, the immune complexes were eluted with SDS sample buffer (Thermo Fisher Scientific) and examined by western blotting. The mouse-derived specific antibody against p150^Glued (BD Biosciences, #610474, 1:1000, recognizing p150^Glued but not p135+) and normal mouse IgG (Santa Cruz, #sc-2025) were used for co-IP.