A commercially available siRNA directed toward the hEGFR was purchased from Dharmacon (SMARTpool Cat. No. L-003114-00, Locus NM_201283; sequence J-003114-10, CAAAGUGUGUAACGGAAUA; J-003114-11, CCAUAAAUGCUACGAAUAU, J-003114-12, GUAACAAGCUCACGCAGUU, J-003114-13, CAGAGGAUGUUCAAUAACU. and siRNA directed toward the mNumb (Santa Cruz Biotechnologies Inc. Cat. No. sc-42147; Locus NM_010949, 110 GUAGCUUCCCAGUUAAGUAtt, 412 CGAUGGAUCUGUCAUUGUUtt, 884 CCCUACGCAUCAAUGAGUUtt). siRNA transfection produced selective knockdown of the hEGFR and mNumb. Briefly, 2μl of 20pM of siRNA solution and 12μl of the transfection reagent were incubated in 100μl of SCM for 20 minutes, in order to facilitate complex formation. The siRNA transfection mix was added to the SVZ cells cultured in 2.5% FBS. Controls consisted of non-specific siRNA. SVZ cells were transfected for 7 hours at 37°C, washed with Hanks’ buffer and cultured in SCM 2.5% FBS for an additional 24 hours. The medium was then changed to basal SCM (20 ng/ml EGF and 10 ng/ml FGF). After 24 or 48 hours, cells were collected and processed for RNA or protein extraction.
