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Fig. 5. SNX9 binds to the Arp2/3 complex via its LC domain. (A) COS-7 cell lysates were immunoprecipitated with anti-Arp3 antibody and immunoblotted (IB) with anti-SNX9 antibody. (B) Schematic diagrams of the GST-tagged truncated LC domains of SNX9 constructs. (C) In vitro binding assays were carried out with purified GST-SNX9 protein fragments (full-length, SH3, LC or PXBAR) and Arp2/3 complex. The bound protein was verified using anti-Arp3 antibody. (D) In vitro binding assays were carried out with various truncated mutant of LC domain and the bound protein was immunoblotted with anti-Arp3 antibody. The VCA domain of N-WASP (GST-VCA) was used as a positive control for Arp2/3 binding. (E) Comparison of the SNX9 A-like domain with the A-like domains of N-WASP, WASP, Scar, Cortactin, Myo1b and ActA. Black box with an asterisk indicates conserved tryptophan residue and gray boxes indicate conserved aspartate or glutamate residues. (F) Endogenous SNX9 and Arp2/3 complex in Cos-7 cells were stained with anti-SNX9 antibody and anti-Arp2/3 antibody, respectively. High-magnification views of the regions enclosed by rectangles are shown below each image. Arrows indicate where two proteins were juxtaposed or overlap. Scale bars: 30 µm (low magnification), 5 µm (high magnification).
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