Fig. 5
From: The NESH/Abi-3-based WAVE2 complex is functionally distinct from the Abi-1-based WAVE2 complex
Analysis of Abi-1/NESH chimeras. (a) Schematic representation of the chimeric proteins. Abi-1 (red) and NESH (blue), and the chimeric proteins used in this study are schematically illustrated. The positions of the WAVE-binding (WAB) domain, the proline-rich region (PXXP motif), the polyproline structure (pps), and the SH3 domain are indicated. (b) FLAG-Abi-1/NESH•SH3 or FLAG-NESH/Abi-1•SH3 was coexpressed with c-Abl in 293T cells. A mouse IgG or an anti-c-Abl antibody was added to lysates of the transfected cells, and the precipitated proteins were analyzed by Western blotting with antibodies against c-Abl and FLAG. To determine the amounts of the proteins expressed, 2 % of each lysate was analyzed (2 % Input). (c) FLAG-chimera N-1 (lane 1), FLAG-chimera N-2 (lane 2), FLAG-chimera N-3 (lane 3), FLAG-chimera N-4 (lane 4), or FLAG-chimera N-5 (lane 5) was coexpressed with c-Abl in 293T cells. c-Abl was immunoprecipitated with the anti-c-Abl antibody, and the precipitated proteins were analyzed as described in (B). (d) 293T cells were cotransfected with expression plasmids for c-Abl and GST-WAVE2, together with an expression plasmid for FLAG (lane 1), FLAG-Abi-1 wild-type (lane 2), FLAG–Abi-1/NESH•SH3 (lane 3), FLAG-NESH wild-type (lane 4), FLAG–NESH/Abi-1•SH3 (lane 5), FLAG-chimera N-1 (lane 6), FLAG-chimera N-2 (lane 7), FLAG-chimera N-3 (lane 8), FLAG-chimera N-4 (lane 9), or FLAG-chimera N-5 (lane 10). The resulting cell lysates were analyzed by Western blotting with each of the antibodies against phosphotyrosine (α-pY), c-Abl, FLAG, and GST. The expression of the wild-type Abi-1 and Abi-1/NESH•SH3 was below the respective detection limit in this assay. However, the phosphorylation of GST-WAVE2 was evident in these cell lysates. The phosphorylated Abi-1 may have been subjected to degradation. (e) NIH3T3 cells expressing Chimera N-1 or Chimera N-5 were plated onto FN-coated coverslips. At the indicated times, the cells were fixed and stained with TRITC-phalloidin (left panels). Cells with lamellipodial structures were counted under a fluorescence microscope. At least 100 cells were analyzed for each sample. Data represent the means ± S.D. for three independent experiments. Error bars represent S.D. (the right graph) (f) FLAG (lane 1), FLAG-Abi-1 (lane 2), FLAG-NESH (lane 3), FLAG-NESH-Abi-1(a.a. 270–336) (lane 4), or FLAG-Abi-1 Y213F (lane 5) was coexpressed with c-Abl in 293T cells. c-Abl was immunoprecipitated with an anti-c-Abl antibody, and the precipitated proteins were analyzed as described in (b). To determine the amounts of the proteins expressed, 2 % of each lysate was analyzed (2 % Input). The expression levels of FLAG-Abi-1 and FLAG-Abi-1 Y213F were low compared with those of FLAG-NESH and FLAG-NESH-Abi-1(a.a. 270–336). However, the precipitation of FLAG-Abi-1 and FLAG-Abi-1 Y213F was evident in these cell lysates. (g) 293T cells were cotransfected with expression plasmids for c-Abl and GST-WAVE2, and together with an expression plasmid for FLAG (lane 1), FLAG-Abi-1 (lane 2), FLAG-NESH (lane 3), FLAG-NESH-Abi-1(a.a. 270–336) (lane 4), or FLAG-Abi-1 Y213F (lane 5). The GST-WAVE2 was pulled down with glutathione beads and analyzed by Western blotting with the antibodies against a phosphotyrosine and GST. To determine the amounts of proteins expressed, 4 % of each lysate was analyzed by Western blotting with the antibodies against c-Abl and FLAG (4 % Input). The expression of FLAG-Abi-1 and FLAG-Abi-1 Y213F was below the detection limit in this assay. However, the phosphorylation of GST-WAVE2 was evident in these cell lysates. (h) The predicted structures of Abi-1 and NESH. The structures of Abi-1 (green) and NESH (cyan) are presented as a ribbon diagram, being maximally superimposed with respect to their SH3 domains