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. 2013 Jul 15;24(14):2216–2227. doi: 10.1091/mbc.E12-12-0883

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© 2013 Yang et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — PakB-1-180 binds the dAbp1 SH3 domain. (A) dAbp1 consists of an ADF-H domain, a segment rich in glutamine, proline, and alanine (GPA) residues, a highly acidic region, and an SH3 domain. The positions of PxxP motifs are indicated by black rings. (B) Two-hybrid analysis was carried out using a bait vector expressing PakB-1-180 (1–180) and a prey vector expressing the dAbp1 SH3 domain (SH3) and empty bait or prey vectors (EV). The strength of the interaction was assessed quantitatively by liquid culture β-galactosidase assay. (C) Pull-down assays were carried out using the GST-dAbp1-SH3 domain (SH3) or an inactive GST-dAbp1-SH3 domain (P474L mutation; SH3(P/L)) and lysates of cells expressing GFP-PakB-1-180. The whole-cell lysate (WCL) and washed pellets were probed using an anti-GFP antibody. Coomassie blue (CB) was used to visualize the GST-SH3 domains in the pellets. (D) Immunoblot analysis of AX3 cells harvested at different stages of development using an affinity-purified rabbit polyclonal antibody to dAbp1. (E) Coimmunoprecipitation of endogenous dAbp1 and PakB. Immunoprecipitates were prepared from AX3 cell lysates using a control (Ctrl) or anti-dAbp1 antibody. The WCL and washed immunoprecipitates were immunoblotted using anti-PakB and anti-dAbp1 antibodies. Results from two experiments are shown. (F) Immunoprecipitates were prepared from lysates of cells expressing GFP-PakB-1-180 (1–180) using an anti-GFP antibody or a control (Ctrl) antibody. The WCL and washed immunoprecipitates were immunoblotted using antibodies to GFP and dAbp1. (G) Immunoprecipitates were prepared from cells expressing GFP-PakB-1-180 (1–180) or GFP-PakB-1-180ΔP (1-180ΔP) using an anti-dAbp1 or a control (Ctrl) antibody. The WCLs and washed immunoprecipitates were immunoblotted using antibodies to GFP and dAbp1.