Fig. 3

Abi-1-based and NESH/Abi-3-based WAVE2 complexes exist independently. (a) Lysates prepared from mouse brain were immunoprecipitated with a mouse IgG, an anti-Abi-1 monoclonal antibody (1G9), or an anti-NESH monoclonal antibody (2H7) (lanes 2 to 4). Lysates prepared from HUVEC were immunoprecipitated with the mouse IgG, the anti-Abi-1 monoclonal antibody (1G9), a rabbit IgG, or a rabbit anti-human NESH antibody as indicated (lanes 6, 7, 9, and 10). The precipitated proteins were analyzed by Western blotting with the indicated antibodies. A rabbit anti-mouse NESH antibody was used to detect NESH in the mouse brain lysates (lanes 1 to 4). The rabbit (lanes 5 to 7) or mouse (lanes 8 to 10) anti-human NESH antibody was used to detect NESH in the HUVEC lysates. (b) FLAG-Abi-1 or FLAG-NESH was coexpressed with GST-Abi-1, GST-NESH, or GST in 293 T cells. GST-Abi constructs were pulled down with glutathione beads, and then the bound proteins were detected by Western blotting with an anti-FLAG or GST antibody (pull-down). To determine the amounts of the proteins expressed, 4 % of each lysate was analyzed (4 % Input)