Figure 1.

EDC with NHS cross-links Acanthamoeba profilin to the Arp2 subunit of the Arp2/3 complex. (A) SDS-PAGE of proteins cross-linked with EDC/NHS and stained with Coomassie blue (cb) or immunoblotted (ib) with monoclonal antiprofilin antibodies. Samples, as indicated by the horizontal bars: 3 μM Arp2/3 complex, Arp2/3 plus 20 μM profilin-II, profilin-II alone. (B) Immunoblot of Arp2/3 complex and profilin-II cross-linked with EDC/NHS and probed with antibodies against profilin (lanes 1 and 4), Arp2 (lanes 2 and 5), and Arp3 (lanes 3 and 6). Lanes 1–3, 3 μM Arp2/3 complex; lanes 4–6, 3 μM Arp2/3 and 20 μM profilin-II. Asterisk marks the two profilin/Arp2 cross-linked products. Identical results were obtained with Acanthamoeba profilin-I. Conditions: Cross-linking was carried out for 1 h at 24°C with 5 mM EDC and 5 mM NHS. Buffer: 150 mM NaCl, 0.2 mM MgCl₂, 0.1 mM EGTA, 0.2 mM ATP, 1 mM DTT, 10 mM imidazole, pH 7.5. Arp2/3 complex was purified by profilin:poly-L-proline affinity chromatography.