Fig. 1.

The shRNA-mediated Abi1 gene silencing. (A) Abi1 expression in Ba/F3 cells (BaF3) and p185^wt cells transduced with either non-silencing shRNA (p185^wt) or Abi1 shRNAs (shRNAs A, B and C). Total lysates from 1 × 10⁶ cells were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to nitrocellulose membrane. The membrane was probed with the antibodies as indicated. The p185 Bcr-Abl and c-Abl were indicated by the arrow and arrowhead, respectively. (B) Efficacy of Abi1 knockdown by different Abi1 shRNAs. The lysates (2 × 10⁷ cells) from the indicated cell lines were immunoprecipitated (IP) by Abi1 antibody and the immunoprecipitates were analyzed by western blotting (WB) using Abi1 antibody as probe (upper panel). Total RNAs from the indicated cell lines were isolated and the complementary DNAs were synthesized. Real-time quantitative PCR (lower panel) was performed to determine Abi1 messenger RNA (mRNA) levels, which are expressed as the levels relative to that of GAPDH. Data represent mean ± SD of triplicate experiments; *P < 0.001. (C) Expression of Abi2 and WAVE2 in Ba/F3 cells and p185^wt cells transduced with either non-silencing shRNA or Abi1 shRNAs. Total lysates from 1 × 10⁶ cells were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to nitrocellulose membrane. The membrane was probed with the antibodies as indicated.