Pull down was performed by using biotin-tagged VCA peptide, MDA-MB-231 breast cancer cell lysate, recombinant VCA Nbs and STREPTactin beads. The two controls (no nanobody (No Nb) or EGFP Nb) show maximal Arp2/3 binding. A concentration range was used (VCA Nb: VCA domain stoichiometry of 0.5x, 1x, 2x, 4x). For each repeat, actin as well as Arp2/3 were analysed on western blot and quantification was done using ImageJ (bars represent mean and SEM, n = 3). Kruskal-Wallis and Duns post test was performed. On top the influence of each VCA Nb on actin—N-WASp binding (A-D) is shown and at the bottom the effect of each VCA Nb on Arp2/3 –N-WASp binding (E-H) is shown.