Fig. 1

Identification of IQGAP1 as a novel Hax1 Binding Partners by Tandem Affinity Purification. A, Hax1 and its associated proteins were isolated by tandem affinity purification and resolved by SDS-PAGE. Immunoglobulin G heavy chain and light chain are marked by arrowheads. The putative band for Hax1 is marked by star. Putative band for IQGAP1 is marked by an arrow. B, IQGAP1 were found as Hax1 binding protein by mass spectrometric sequencing of proteins from the coomassie blue-stained gel. Identified matched peptides were shown in bold red. C, HEK293T cells were transfected with different plasmids as indicated. Cell lysates were immunoprecipitated (IP) with HA-agarose beads and immunoblotted (IB) with different antibodies as indicated. For whole-cell lysate (WCL), 10 μg of total protein was used. Note that only Hax1 specifically pulls down IQGAP1. D, Hax1 immunoprecipitates from MCF7 cells were immunoblotted with antibodies against IQGAP and Hax1. For whole-cell lysate (WCL), 10 μg of total protein was used. Note that Hax1 immunoprecipitation brought down IQGAP1. E, MCF7 cells were immunostained for IQGAP1 (red), Hax1 (green), and DAPI (blue). Scale bar, 10 μm. Cells were imaged by Nikon's Structured Illumination microscope. The boxed areas are magnified as insets. Note that Hax1 colocalizes with IQGAP1 signal in the protrusive region and accumulates at the leading edge of migrating cells