Figure 13.

EDC cross-links actin to Arp3, to p35, and to one of the low molecular weight subunits of the Arp2/3 complex. Conditions: Cross-linking was carried out for 1 h at 24°C with 2 μM purified Arp2/3 complex, 4 μM filamentous actin, 5 mM EDC, 5 mM NHS, 10% DMSO, 50 mM KCl, 0.2 mM MgCl₂, 0.1 mM EGTA, 10 mM imidazole, pH 7.5. Reaction was quenched with an equal volume of 1 M Tris-HCl, pH 8.0, and the products analyzed by SDS-PAGE and immunoblotting. Immunoblots were probed with monospecific antibodies against Arp3, p35, and actin at dilutions of 1:12,000, 1:6,700, and 1:10,000, respectively. Purified Arp2/3 complex and Acanthamoeba actin were incubated with EDC and NHS, and the reactions were quenched. (A) Immunoblots of EDC/NHS-cross-linked samples probed with monoclonal antiactin antibodies. Lane 1, EDC-treated actin filaments; lane 2, EDC-treated mixture of actin filaments and Arp2/3 complex showing three prominent new bands at (i) 97, (ii) 79, and (iii) 59 kD. (B) Immunoblots of EDC/NHS-cross-linked samples probed with antibodies against p35 (lanes 1 and 2), actin (lane 3), or Arp3 (lanes 4 and 5). The cross-linking reactions contained Arp2/3 complex, actin filaments, or both as indicated with +. Arrows denote new bands recognized by the anticomplex antibodies that correspond to new bands recognized by antiactin antibodies.