The barbed end assay was performed using biotin-conjugated actin as previously described [72 (link)]. In brief, cells were starved, stimulated with 2.5 nM EGF and permeabilized with a permeabilization buffer (20 mM Hepes, pH 7.5, 138 mM KCl, 4 mM MgCl2, 3 mM EGTA, 0.2 mg/ml saponin, 1 mM ATP, and 1% BSA) containing 0.4 μM biotin-conjugated muscle actin (AB07, Cytoskeleton Inc.) for 1 minute at 37° C. Cells were fixed in 3.7% paraformaldehyde for 5 minutes, blocked in PBS containing 1% FBS, 1% BSA and 3 μM un-labeled phalloidin (Molecular Probes, P3457), then labeled with FITC anti-biotin (200-092-211, Jackson ImmunoResearch Laboratories) to visualize barbed ends, and with rhodamine-phalloidin (Molecular Probes, R415) and Arp2 to identify regions of cells rich in invadopodia. To image newly formed barbed ends, which form at invadopodia at the ventral side of the cell, we first focused on Arp2 and actin which are localized at the cell leading edge. We then analyze only the pixels corresponding to invadopodia dots.
The barbed end intensity at invadopodia-rich regions was quantified by measuring mgv at invadopodia-rich regions minus mgv of the background. Data was normalized to the control condition for each experiment.
The barbed end intensity at invadopodia-rich regions was quantified by measuring mgv at invadopodia-rich regions minus mgv of the background. Data was normalized to the control condition for each experiment.