FIG. 3.
ANTXR1-sv1 associates with the actin cytoskeleton. (A) U20S cells were stably transfected with ANTXR1-sv1-HA, ANTXR1-sv2-HA, or pcDNA3-HA. Cells were lysed, and the lysates were mixed with anti-HA antibody. Antibody and associated proteins were precipitated with protein A-Sepharose, subjected to SDS-PAGE, and stained with silver. The asterisk indicates an ∼45-kDa protein that coimmunoprecipitates with ANTXR1-sv1-HA but not with ANTXR1-sv2-HA or HA alone. (B) FRAP curves for CHOR1.1 cells expressing either ANTXR1-sv1-EGFP (red squares) or ANTXR1-sv2-EGFP (blue triangles) and treated with either latrunculin A (right panel) or DMSO vehicle (left panel). The data were averaged over seven independent experiments. Error bars represent standard errors of the means. (C) CHOR1.1 cells or cells expressing ANTXR1-sv1-HA or ANTXR1-sv2-HA were subjected to 0.15% Triton X-100 extraction for 5 min on ice and centrifuged for 30 min at 4°C. Proteins that fractionated in the pellet (P) or supernatant (S) were subjected to Western blotting with anti-HA and anti-β-actin antibodies. (D) Cells expressing ANTXR1-sv1 were pretreated with latrunculin A or DMSO vehicle for 45 min at 37°C prior to Triton X-100 extraction as described for panel C.