C abl sirna

Detail of this protocol was first published in [27 (link)]. In brief, HME and Gem9 cells were incubated in growth factor-free medium for 72 hours to synchronize cells in G₀/G₁ phase (> 95%) G₀/G₁ cells were then released from arrest in medium containing growth factors, and 16 hours (S phase), 22 hours (G₂/M phase) or 24 hours (M/G₁ phase) later cells were collected and analyzed. For more information regarding this protocol [27 (link), 51 (link)]. Cell transfection with double-stranded RNA interference (siRNA) reagent was performed as described by [51 (link)]. In brief, cells were transfected (0 hours) in serum-free medium with a relevant by a standard method using oligofectamine. At 24 hours, the medium was changed, and growth factor-containing MEBM (Clonetics/Cambrex, Waltersville, MD) was added. Small interfering RNA (siRNA) used were siGem: TGAGCTGTCCGCAGGCTTT, scrambled siGem: TGATTTGTCCGCAGCTGGC and c-Abl siRNA was premade from Dharmacon. The silenced luciferase (siLuc) was from previously published data. For more information see [24 (link)–27 (link)].

Geminin siRNA was described in [8] (link). A pre-synthesized c-Abl siRNA from (Dharmacon) was used. Transfection of siRNAs in breast cancer as well as HME cells was performed using Oligofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Shcontrol/ and shc-Abl/GFP was a kind gift from Dr. Lindsey D. Mayo (Wells Center for Pediatric Research, Indianapolis).