Abi1 and p130Cas silencing alter Bcr-Abl/C3G interaction. (A) Western blot analysis of the expression of Abi1, Cbl, p130Cas and p87C3G in whole cell lysates from K562 clones stably transfected with the indicated lentiviral shRNA particles. Relative Abi1/β-tubulin, Cbl/β-tubulin, p130Cas/β-tubulin and C3G/β-tubulin ratios are shown. shAbi1-2, shCbl-1, shp130Cas-5, shC3G-1 and shC3G-8 were selected as representative knock-down clones in the experiments. C: whole cell lysate of a K562 clone expressing control lentiviral shRNA. Pull-down assays with lysates of K562 shAbi1-2, shCbl-1 and shControl (shCT) clones (B) or K562 shp130Cas-5 and shCT clones (C), using Abl-SH3 domain fused to GST as bait. p140C3G, p87C3G, Abi1, CrkL, Cbl and p130Cas expression was detected with specific antibodies. Panels showing p140C3G and p87C3G in Figure 3B correspond to different exposure times (longer for p140 and shorter for p87) for a better visualization of both isoforms. Beads with lysis buffer (B), GST construct with K562 whole cell lysate (GST) and K562 whole cell lysate with lysis buffer (LB) were used as negative controls. L: whole cell lysate (40 μg).