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. 2007 Oct 17;104(43):17117–17122. doi: 10.1073/pnas.0703196104

Fig. 5.

Fig. 5.

rVopL enhances actin filament assembly in vitro. (A) A rVopL (0.1–5 nM) or rVopL-WH2×3* mutant was incubated with 4 μM actin (5% pyrene-labeled), and changes in fluorescence were measured over time. (B) Comparison of rVopL (5 nM) and Arp2/3 (5 nM) maximally activated with WASP VCA (500 nM). The proteins were incubated with 4 μM actin (5% pyrene-labeled), and changes in fluorescence were measured over time. “Control” refers to the spontaneous assembly of 4 μM actin (5% pyrene-labeled). (C) VopL-nucleated filaments grow at their barbed ends. Shown are pyrene actin polymerization assays in the presence of actin (0.5 μM 20% pyrene), 5 nM VopL-WH2C (WH2C), and mouse capping protein α1β2 (CP) from 0.2 nM to 10 nM. At 10 nM, CP completely inhibits barbed-end filament growth. (D) VopL has no effect on filament elongation. Shown is elongation of 1 μM phalloidin-stabilized actin filaments with 0.5 μM actin monomer (40% pyrene) in the presence and absence of 3 nM VopL-WH2C (WH2C). (E) VopL binds filament sides. Shown are filament binding assays with GST-VopL-WH2C (GST-WH2C) and 5 μM phalloidin-stabilized actin filaments. In this assay, GST-VopL-WH2C is used so that it can be distinguished from actin by SDS/PAGE. Lane 1, 1 μM GST-VopL-WH2C alone; lanes 2–6, 5 μM actin filaments and 1, 2.5, 5, 7.5, and 10 μM GST-VopL-WH2C, respectively; lane 7, 5 μM actin filaments and 10 μM GST; lane 8, 5 μM actin filaments and 10 μM CP. (F) Coomassie-stained SDS/PAGE gel of rVopL (wt, 1 μg) and rVopL-WH2×3* (mt, 1 μg).