Immunoblot Analysis of Cellular Proteins

Immunoblot analysis was carried out as previously described with slight modifications^{12 (link)}. Briefly, cell lysates were prepared in immunoprecipitation (IP) buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1% NP-40, 1% sodium deoxycholate with 1 mM PMSF, 1 μg/μl aprotinin, and 1 μg/μl leupeptin as protease inhibitors) and incubated on ice for 30 min. Total cell lysates (15 μg) were subjected to SDS-PAGE, followed by immunoblot detection with anti-Tuj1 (1:1,000, Millipore), anti-Ub (1:1,000, Millipore), anti-Notch3 (1:200, Santa Cruz Biotechnology), anti-CC3 (1:1,000, Millipore) or anti-β-Actin antibody (1:2,000, Santa Cruz Biotechnology). Based on the types of primary antibodies, the appropriate HRP-conjugated goat anti-mouse or anti-rabbit IgG (1:10,000, Enzo Life Sciences) or HRP-conjugated donkey anti-goat IgG (1:5,000, Santa Cruz Biotechnology) was used.