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. Author manuscript; available in PMC: 2010 Nov 13.
Published in final edited form as: Mol Cell. 2009 Nov 13;36(3):512. doi: 10.1016/j.molcel.2009.10.024

Figure 1. Native WAVE2 and WAVE1 complexes are basally inactive and are not activated by Rac or Nck.

Figure 1

(A) Scheme used to purify native WAVE2 complex from pig leukocyte extract and native WAVE1 complex from bovine brain extract. The sucrose gradient step indicated in brackets was only included during purification of the WAVE1 complex.

(B) Native WAVE2 complex from pig leukocytes and native WAVE1 complex from bovine brain, resolved on 4–12% Bis-Tris polyacrylamide gels and stained with Coomassie (top panels) or silver (bottom panels). Constituent subunits, identified previously by mass spectrometry, are indicated.

(C–G) The activity of WAVE complexes was tested in polymerization assays containing 1 μM actin/pyrene-actin and 30 nM Arp2/3 complex (C–F) or 2 μM actin/pyrene-actin and 60 nM Arp2/3 complex (G).

(C) Native WAVE2 complex does not stimulate actin polymerization. The indicated concentrations of WAVE2 complex from pig leukocytes (“WAVE2c”) or isolated recombinant WAVE2 protein (“rWAVE2”) were tested. Recombinant WAVE2 protein activates the Arp2/3 complex as expected.

(D) Native WAVE2 complex from pig leukocytes is not activated by Rac2 or Nck. 200 nM constitutively active prenylated Rac2-GTP (“pRac2-GTP”), 200 nM full length Nck or both were added to reactions containing 40 nM WAVE2 complex (“WAVE2c”) as indicated. A reaction containing 16 nM recombinant WAVE2 protein (“rWAVE2”) is shown for reference.

(E) Thermal denaturation activates the WAVE2 complex. WAVE2 complex from pig leukocytes was heated for 10 minutes at the indicated temperature, cooled down to room temperature and 40 nM was immediately added to the assay.

(F) Native WAVE1 complex is inactive and is not activated by Rac1 or Nck. 20 nM WAVE1 complex from bovine brain (“WAVE1c”) was tested alone or with 200 nM constitutively active unprenylated Rac1-GTP (“uRac1-GTP”), 200 nM full length Nck, or both. Thermal denaturation (57°C) revealed the activity of the WAVE1 complex.

(G) Freezing and thawing without cryoprotectants activates the WAVE1 complex. WAVE1 complex from bovine brain was exchanged into buffer XB, freeze thawed directly or after addition of 10% glycerol, and assayed at a final concentration of 20 nM.

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