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. 2012 Nov 26;288(2):1184–1199. doi: 10.1074/jbc.M112.431825

FIGURE 1.

FIGURE 1.

ELMO proteins directly interact with ACF7. A, schematic representation of ELMO1 and ACF7 constructs used in this study. B, a spectrin repeat of ACF7 interacts with the C terminus of ELMO1. Yeasts transformed with LexA fusion constructs of ELMO1 together with a B42 fusion construct of ACF7 were grown in nonselective (−histidine, −tryptophan) and selective (−histidine, −tryptophan, −leucine) conditions. C, ELMO1 co-immunoprecipitates with ACF7. Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation using anti-GFP (ACF7) antibody. Immunoblots were analyzed with anti-Myc (ELMO1) and anti-GFP (ACF7) antibodies. D, endogenous ACF7 and ELMO co-localize at the cell cortex. MDA-MB-231 cells were allowed to spread on fibronectin. Fixed cells were stained with a rabbit polyclonal antibody recognizing ACF7, a goat polyclonal antibody against ELMO and DAPI. Scale bars, 10 μm. E, ACF7 interacts with ELMO proteins. Lysates of HEK293T cells transfected with GST alone or GST-ACF74700–4945 together with ELMO1, ELMO2, or ELMO3 were subjected to GST pulldown assays. Immunoblots were analyzed using anti-Myc (ELMO proteins) and anti-GST (GST or GST-ACF74700–4945) antibodies. F, ACF7 interacts with the polyproline region of ELMO1. Lysates of HEK293T cells transfected with GST alone or GST-ACF74700–4945 together with ELMO1, ELMO11–315, ELMO1315–727, or ELMO1PxxP were subjected to GST pulldown assays. Immunoblots were analyzed using anti-Myc (ELMO1) and anti-GST (GST or GST-ACF74700–4945) antibodies. TCL, total cell lysate.