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Fig. 7. Expression of Abi1^PPLL inhibited Bcr-Abl-induced abnormal actin cytoskeleton remodeling. (A) Schematic diagram of Abi1 and Abi1^PPLL. (B) Abi1^PPLL is defective in binding to Bcr-Abl. Ba/F3 cells (lane 1) and the Ba/F3 cells expressing p185^wt alone (lane 2), p185^wt plus HA-tagged Abi1 (lane 3) and p185^wt plus HA-tagged Abi1^PPLL (lane 4) were lysed and immunoprecipitated with anti-Abl antibody. The immunoprecipitates (middle and lower panels) and 1/50 of total cell lysates used for immunoprecipitation (IP) (Input, upper panel) were analyzed by western blot using indicated antibodies. The HA-tagged Abi1 and Abi1^PPLL are indicated by arrowheads, whereas a non-specific band crossreacted with anti-HA antibody is indicated by the arrow (upper panel). The anti-Abl antibody recognized both Bcr-Abl and endogenous c-Abl, as indicated by arrowhead and arrow, respectively (middle panel). (C) Abi1^PPLL failed to be tyrosine-phosphorylated by Bcr-Abl. Ba/F3 cells (lane 2) and Ba/F3 cells expressing p185^wt alone (lane 1) or p185^wt plus HA-tagged Abi1^PPLL (lane 3) were lysed and immunoprecipitated with indicated antibodies. The immunoprecipitates were analyzed by western blot using indicated antibodies. (D) Inhibition of Bcr-Abl-induced abnormal actin remodeling by Abi1^PPLL. Ba/F3 cells transformed by p185^wt were transduced with retroviruses expressing either GFP-Abi1 (a-c) or GFP-Abi1^PPLL (d-f). Cells were fixed and stained with TRITC-conjugated phalloidin and DAPI to visualize F-actin (red) and nuclei (blue), respectively. Subcellular localization of GFP-fusion proteins (b,e, green) and F-actin structure (a,d, red) were analyzed by two-photon confocal microscopy. The merged images were also shown (c,f). The arrowheads in panel c indicate abnormal actin-enriched structures that colocalize with GFP-Abi1, whereas arrows in panel f indicate the p185^wt-transformed cells that express GFP-Abi1^PPLL. An open arrowhead in panel f indicates abnormal actin-enriched structure in a cell that did not express Abi1^PPLL. Bar, 10 µm.

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