Sc 27046

Embryos were lysed in buffer (50 mM Tris, pH 7.5, 5% glycerol, 0.2% Igepal, 1.5 mM MgCl₂, 125 mM NaCl, 25 mM NaF, 1 mM Na₃VO₄, 1 mM dithiothreitol, supplemented with Complete Protease Inhibitor cocktail with or without EDTA [Roche, Indianapolis, IN]). For collection of neural/axonal lysates, neural tube explants were grown on coverslips overnight. Medium was then removed and replaced with lysis buffer (as described), and cultures were scraped to obtain the neural tissue. Blotting was carried out using goat anti–X. laevis TACC3 (1:500; sc-27046; Santa Cruz Biotechnology, Dallas, TX) and rabbit anti-XMAP215 (1:2500; a gift from the Hyman lab). Mouse anti–β-actin (1:2500; ab8224; Abcam) was used as a loading control. For TACC3 detection, 1% immunoglobulin G (IgG)-free bovine serum albumin (BSA) in phosphate-buffered saline/Tween-20 (PBST) was used to block nitrocellulose membrane. For other antibodies, 2% nonfat milk was used. Detection was done by chemiluminescence using Amersham ECL Western Blot reagent (GE Healthcare, Pittsburgh, PA). The bands were quantified by densitometry using Photoshop (Adobe, San Jose, CA), and the data and graphs were compiled in Excel (Microsoft, Redmond, WA).

Embryonic explant cultures were fixed in 100% cold methanol for 3 min and then rinsed in PBST and blocked in 0.1% Triton-X and 1% IgG-free BSA in PBS before antibody incubation. Immunostaining was carried out using goat anti-TACC3 (1:1000; sc-27046; Santa Cruz Biotechnology) and mouse anti-tubulin DM1α (1:1000; Sigma-Aldrich, St. Louis, MO). Donkey anti-goat Alexa Fluor 488 and donkey anti-mouse Alexa Fluor 568 antibodies (1:1000; Life Technologies) were used as secondary antibodies.