Rabbit anti β tubulin 9f3

Brains were dissected from P1 Ephb1 knockout, heterozygous and wild-type mice and lysed in RIPA buffer (150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM Tris-HCl, pH 8) with proteinase inhibitors (S8830, Sigma-Aldrich). Mouse ES cells were treated with 2 μg/ml doxycycline for 48 h and harvested in RIPA buffer. After quantification by Bio-Rad protein assay (Bio-Rad), proteins were resolved in 4–12% SDS-PAGE gels and transferred to a nitrocellulose membrane. The western blot was performed using goat anti-EphB1 (M19, Santa Cruz, 1:250), rabbit anti-Fezf2/Fezf1 (F441, IBL, 1:500), rabbit anti-β-tubulin (9F3, Cell Signaling, 1:500) and mouse anti-α-tubulin (T9026, Sigma, 1:5,000). Anti-goat, anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (HRP-linked anti-rabbit IgG, Cell Signaling Technology 7074, HRP-conjugated goat anti-mouse IgG, Cell Signaling Technology 7076, HRP-conjugated anti-goat IgG, Santa Cruz sc-2020, all at 1:5000), followed by ECL (Amersham), were used to visualize proteins of interest on the membrane.