For immunoprecipitation, SVZ tissue extracts from WT and CNP-hEGFR mice were prepared in RIPA buffer containing. Aliquots (200μg protein) were incubated overnight with antibodies against ubiquitin (Santa Cruz Biotechnology), Numb or Notch1 and 15μl of Agarose A (Santa Cruz Biotechnology). Immunocomplexes bound to agarose A were collected by centrifugation and washed twice in 500μl RIPA buffer containing inhibitors. Precipitated proteins were analyzed by immunoblotting. Bands were detected using HRP and developed with a chemiluminescent substrate (ECL, Amersham).
