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Fig. 3. SCAR knockdown in S2R+ cells. (A) Morphological phenotype developed after SCAR-specific RNAi. Cell spreading is compromised; numerous long processes appear along cell edges. (B) Immunoblotting. Extracts of S2R+ cells subjected to SCAR RNAi for 0-4 days (lanes 0-4, respectively) were probed with antibodies to SCAR or WASp. Lane 4+5 WO shows restoration of SCAR level after washout of dsRNA on day 4 and culturing of cells for an additional 5 days under regular conditions. Equal load of protein was ensured by tubulin probing (not shown). Positions of markers are indicated. Reduction of SCAR is >95% by day 4 of treatment. (C) RT-PCR after Laser Capture Microdissection (LCM). 100 control cells (lanes labeled `–') and 100 cells subjected to SCAR RNAi for 4 days (lanes labeled `+') were captured by LCM (see text for details) and analyzed by RT-PCR using SCAR or rp49 specific primers. Reduction of SCAR mRNA is 95% by densitometry; rp49 mRNA serves as an internal control, and its levels do not change. The positions of DNA markers and number of PCR cycles are indicated. (D) Immunostaining. Control cells (top row) and cells treated with dsSCAR for 4 days (bottom row) were stained with phalloidin to reveal actin (left panels) and with SCAR antibody (middle). Merged images (right) show that in control cells SCAR is enriched at the leading edge of lamellipodium (arrow), and in dsSCAR-treated cells, SCAR is not detected in peripheral protrusions. Bars, 10 µm (A), 2 µm (D).

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