XB-IMG-136642
Xenbase Image ID: 136642
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Figure 3. The interaction between Lpd and the Scar/WAVE complex is mediated by the Abi SH3 domain and positively regulated by active Rac. (A–D) The Abi SH3 domain and three Abi binding sites in Lpd mediate the interaction between Lpd and the Scar/WAVE complex. Immunoprecipitation using Lpd antibodies or IgG control from HEK293 cell lysates (A) expressing GFP-Lpd and all Myc-tagged components of the Scar/WAVE complex, including Myc-Abi1full-length (A, left), Myc-Abi1ΔSH3 (A, right), or GFP-Abi and GFP-Lpd (C, left) or GFP-LpdAbiMut (C, right), show coimmunoprecipitation between Lpd and all components of the Scar/WAVE complex only when the Abi SH3 domain is present (A; Myc-HSPC300 is not shown) or between Lpd and GFP-Abi only when the Abi binding sites are present (B). Western blot: anti-GFP. (B and D) Comparison of efficiency of coimmunoprecipitation of Lpd with all components of the Scar/WAVE complex (B) or GFP-Lpd or GFP-LpdAbiMut with Abi (D). Quantification of band intensity of chemiluminescence imaged with a charge-coupled device camera. (B) Coimmunoprecipitation is reduced by >90%. Error bars indicate mean ± SEM, n = 3. One-way analysis of variance (ANOVA) and Tukey’s test were used; **, P < 0.01. (D) Coimmunoprecipitation is reduced by >94%. Error bars indicate mean ± SEM, n = 3. An unpaired t test was used; ***, P < 0.001. (E and F) Lpd and the RA-PH domains of Lpd are in complex with active Rac. Purified GTPγS- or GDPβS-loaded GST-Rac, or GST only as control, on Sepharose beads were incubated with lysates from HEK cells expressing GFP-Lpd (E) or GFP-Lpd-RAPH (F) and bound with GFP-Lpd or GFP-Lpd-RAPH. Samples were detected in a Western blot against GFP. (G) The RA-PH domains of Lpd directly interact with active Rac. Purified GTPγS- or GDPβS-loaded GST-Rac or GST only as control Sepharose beads were incubated with MBP-Lpd-RAPH or MBP only as control, and direct interaction was detected in a Western blot against MBP. (H and I) The interaction between Lpd and Abi is positively regulated by active Rac. Immunoprecipitation using Lpd-specific antibodies or IgG control from HEK293 cell lysates expressing GFP-Abi, GFP-Lpd, and dominant-active Rac (DA Rac; H, left) or dominant-negative Rac (DN Rac; H, middle), or empty vector control (H, right) show increased coimmunoprecipitation between Lpd and GFP-Abi only when dominant-active Rac is coexpressed. Western blot: anti-GFP. (I) Comparison of efficiency of coimmunoprecipitation of Lpd with GFP-Abi from blots in H. Quantification of band intensity of chemiluminescence imaged with a charge-coupled device camera. Coimmunoprecipitation is increased by >100% compared with empty vector and 150% compared with DN-Rac. Error bars indicate mean ± SEM, n = 3. One-way ANOVA and Tukey’s test were used. *, P < 0.05; **, P < 0.01. Image published in: Law AL et al. (2013) © 2013 Law et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View |