FIG. 6.

ANTXR1-sv1-Y383C binds more PA than ANTXR1-sv1 does and does not associate with the actin cytoskeleton. (A) CHOR1.1 cells or cells transiently expressing ANTXR2⁴⁸⁹-HA or the JHF-linked mutant, ANTXR2⁴⁸⁹-Y381C-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. (B) CHOR1.1 cells or cells expressing ANTXR1-sv1-HA or ANTXR1-sv1-Y383C-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. (C) Receptor-negative CHOR1.1 cells or CHOR1.1 cells transiently transfected with either ANTXR1-sv1-HA or ANTXR1-sv1-Y383C-HA were treated with a non-membrane-permeative biotinylation reagent. Biotinylated proteins were precipitated with streptavidin-agarose and analyzed by Western blotting using anti-HA antibody. (D) Cells were incubated with PA_SSSR for 2 h on ice. The cells were then washed with PBS and lysed, and the lysates were subjected to Western blotting with anti-PA antibody. Blots were also probed for β-actin to ensure equal loading. Blots are representative of three independent experiments.