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Files in this Data Supplement:
Fig. S1. Phosphorylation of WAVE2 does not affect its ability to stimulate the Arp2/3 complex. FLAG-tagged WAVE2 was overexpressed in Cos-7 cells and purified by immunoprecipitation with anti-FLAG(M2) beads. Protein isolated in this way contains a high degree of phosphorylation (see Figs 1 and 2). WAVE2 on beads was further treated with recombinant Erk2 and Jnk2 for 30 minutes, as described in Materials and methods. Immunoprecipitates were washed thoroughly and eluted with FLAG peptide (Sigma), and dialysed against 10 mM Tris pH 8.0. The protein concentration was estimated by the Bradford assay. The Arp2/3 complex was purified from platelets as described previously (Welch and Mitchison, 1998). Actin was purified from rabbit skeletal muscle and pyrene actin purchased from Cytoskeleton. Polymerisation assays were performed in 50 mM KCl with 10 nM Arp2/3 complex and 2.5 μM actin (0.125 μM) pyrene actin. Fluorescence was measured using excitation of 365 nm and emission at 405 nm. Note that spikes observed in some data traces are anomalies and were not repeatable.
Fig. S2. WAVE2 knockdown does not affect ruffling in response to PDGF. NIH 3T3 cells were treated with control siRNA or siRNA targeting WAVE2 (WAVE2 KD). Cells were plated on fibronectin-coated glass coverslips overnight in 0.5% serum. PDGF was added where indicated for 25 minutes before the cells were fixed, permeabilised and stained with phalloidin (F-actin). Bar, 30 μm. (B) Six random microscopic fields were imaged and the number of cells with one or more ruffles were counted. Dark bars (+) represent cells with ruffles, light-grey bars (−) represent those without ruffles. A statistical analysis with Fisher's exact test revealed no significant differences between cell lines in either starved or stimulated conditions.
Fig. S3. WAVE2 localisation in NIH 3T3 fibroblasts. Representative images of GFP-WAVE2 (W2) localisation in NIH 3T3 cells. Cells were grown on glass coverslips in growth medium containing 10% serum, fixed with 4% formaldehyde and stained with phalloidin before imaging. Images represent the compression of five confocal stacks through the cells. Bar, 15 μm.
Fig. S4. Analysis of members of the WAVE complex following knockdown of WAVE2 in EGFP-WAVE2 cell lines. Cell lines expressing EGFP alone, EGFP-WAVE2 WT (W2-WT) or EGFP-WAVE2 AAA (W2-AAA) were treated with either control siRNA (C) or WAVE2 siRNA (KD) and the lysates analysed by immunoblotting with the indicated antibodies.
Movies 1-6. Time-lapse imaging of wound healing. Stable cell lines expressing either EGFP (Movies 1, 4), EGFP-WT WAVE2 (Movies 2, 5) or EGFP-AAA WAVE2 (Movies 3, 6) were treated with control siRNA (Movies 1-3) or WAVE2 siRNA (Movies 4-6) to knockdown the levels of endogenous WAVE2 protein. Images were taken every 30 minutes for 16 hours.
Movies 7-9. Stable cell lines expressing either EGFP (Movie 7), EGFP-WT WAVE2 (Movie 8) or EGFP-AAA WAVE2 (Movie 9) were plated on glass coated with fibronectin (25 μg/ml), left overnight and wounded in medium containing 10% serum. Frames were taken every 6 minutes for 3 hours, with the first frame taken 10 minutes after wounding.
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