SVZ tissue (WT and CNP-hEGFR mice) was micro-dissected from 300μm-thick coronal sections of P8 and P90 brains, and used for protein extraction using lysis buffer (50mM Tris-HCl, pH7.5, 1mM EDTA, 1mM EGTA, 1mM sodium orthovanadate, 50 mM sodium fluoride, 0.1% 2-mercaptoethanol, 1% triton X-100, plus proteases inhibitor cocktail; SIGMA). Protein samples (20μg) were separated on GENE Mate express Gels 420% (ISC BioExpress, Kaysville, UT) and transferred to PVDF membranes (Millipore, Bedford MA). Numb, Notch1, NICD, Dll1, Hes1, RBPjk, ubiquitin, and actin proteins, were detected using an enhanced chemiluminescence substrate mixture (ECL Plus, Amersham, Buckinghamshire, UK). Selective primary antibodies from Santa Cruz Biotechnologies (Caramillo CA) were used at 1μg/ml. Antibodies were used in combination with a secondary horseradish peroxidase-conjugate (Santa Cruz Biotechnologies).
