FIGURE 3:

β1 integrin interacts with Arg in invadopodia. (A) Representative Western blot from the Arg bead pull-down assay. MDA-MB-231 cell lysates were incubated with BSA (negative control) or full-length Arg-coated beads, and the resulting pull-downs were run on SDS–PAGE and stained for β1 integrin. Two independent experiments. (B, C) β1 integrin–Arg acceptor photobleaching FRET. (B) Representative images of FRET efficiency between β1 integrin and Arg at Tks5-rich mature invadopodia in MDA-MB-231 cells at steady state (s.s.) or after integrin activation with 1 mM MnCl₂ for 30 min (Mn²⁺). Dashed white circles denote mature invadopodia. Inset, magnified view of β1 integrin and Arg colocalized at invadopodia and the associated FRET efficiency. LUT bar indicates linear scale of FRET efficiency from 0 to 16%. Bar, 10 μm. (C) Quantification of β1 integrin–Arg and β1 integrin–cortactin FRET efficiencies at invadopodia. n > 61 invadopodia; n > 23 cells; three independent experiments. *p < 0.003. (D) Principle of PLA. Cells are fixed, permeabilized, and stained with primary antibodies (proteins of interest depicted as red and blue spheres). Secondary antibodies conjugated to complementary oligonucleotides are added. The oligonucleotides are hybridized and ligated, and the circular DNA is replicated by rolling circle amplification, incorporating fluorescently labeled nucleotides that can be detected by microscopy. Adapted from Olink. (E) Representative maximum-intensity Z‑projection images of MDA-MB-231 cells showing colocalization of β1 integrin-–Arg PLA events (green) with mature, Tks5-rich invadopodia (red; arrowheads) and negative control β1 integrin–IgG PLA events, which almost never colocalize with mature invadopodia. (F) Quantification of PLA signal colocalization with mature invadopodia. n > 131 invadopodia; n > 62 cells; three independent experiments. *p < 9.34E-5.