Compounds were purchased from Chemdiv, San Diego CA: CK-0944636 (Catalog number 8012-5103), CK-0993548 (Catalog number K205-1650), CK-0944666 (Catalog number 8012-5153) and CK-0157869 (Catalog number K205-0942). We purified native Arp2/3 complex from human platelets12 (link), bovine thymus6 (link), Schizosaccharomyces pombe13 (link) and Saccharomyces cerevisiae (Supplemental methods), actin from chicken skeletal muscle14 (link) and recombinant HsWASp, WASp105-502, WASp-VCA and Cdc4212 (link), N-WASp-VCA 428-505 (Supplemental methods), GST-ActA 36-170 (Supplemental methods) and S. pombe Cdc12p(FH2)-His 973-139015 (link) from E. coli. We used standard assays to measure polymerization of pyrenyl-actin16 (link) and to visualize actin filaments by fluorescence microscopy17 (link). Binding of etheno-ATP to Arp2/3 complex was performed as described previously with slight modifications18 (link). We crystallized BtArp2/3 complex7 (link) with either 0.5 mM CK-548 or 1 mM CK-636 in DMSO or soaked these compounds into crystals for 24 hours before freezing in liquid nitrogen. Diffraction data were collected at beamline X29A at Brookhaven National Laboratories. SKOV3 cells were infected with Listeria monocytogenes and fixed with 2% formaldehyde, permeabilized with 0.1% Triton-X in PBS, stained with Listeria antibody (US Biologics, Cleveland, Ohio) and Alexa Fluor 568 phalloidin (Molecular Probes, Eugene, OR), and imaged by fluorescence microscopy. We used an Isodata threshold on background-subtracted images of Listeria to isolate individual bacterium and measure the ratio of colocalized actin to Listeria fluorescence. Monocyte THP-1 cells were differentiated in 50 nM phorbol myristate acetate (Sigma-Aldrich-Fluka) to form podosomes before treatment with compounds. Black molly keratocytes19 (link) were observed by time-lapse phase contrast microscopy.