Rabbit pab to 14 3 3ζ

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.

Mouse cortical neurons (14 DIV) were fixed with cold methanol for 20 min at −20 °C and permeabilized with 0.2% Triton X-100 in Tris-buffered saline for 5 min. The neurons were stained with the indicated combinations of primary antibodies for 60 min at RT. Staining patterns were visualized by incubating with the Alexa Fluor 488-, Alexa Fluor 594- or Alexa Fluor 647-conjugated donkey secondary antibodies (1:500; Invitrogen) for 60 min at RT. The cell images were obtained with an LSM510META laser scanning confocal microscope (Zeiss). For immunofluorescence of 14-3-3ζ/θ and dynactin1, the following antibodies were used: rabbit pAb to 14-3-3ζ (1:50; Santa Cruz Biotechnology); rabbit pAb to 14-3-3θ (1:50; Santa Cruz Biotechnology); and mouse mAb to dynactin1 (1:250; Transduction Laboratories). For double immunofluorescent staining of γ2 and early endosome antigen 1 in Supplementary Fig. 3g, mouse mAb to γ2 (1:500; Synaptic Systems) and rabbit pAb to early endosome antigen 1 (1:100; Cell Signaling Technology) were used. Cell nuclei were visualized with TO-PRO-3 iodide (1:10,000; Invitrogen). Quantitative analyses of the colocalization were performed using ImageJ, based on the previously reported method42 (link).