Additional file 4.
Study of siRNA efficiency. A. Arp3 knockdown affects cell morphology. HeLa cells were transfected with indicated siRNA for 72 hours, permeabilised and fixed as previously indicated and treated for indirect Alexa 555 localisation of Arp3 (a, e and i) using specific antibody. F-actin was stained with FITC-coupled phalloidin (b, f and j). Merged images are presented (c, g and k). Exposure times for Alexa 555 and FITC channels were determined for control conditions and applied to N-WASP and Arp3 null cells allowing direct comparison of protein levels based on signal intensities. Arp3 localised at the tip of membrane protrusion (a and e, arrowhead) and in vesicle-like structures where it colocalised with F-actin (c and g). Arp3 staining is greatly reduced in Arp3 null-cells (compare i with a and e). Bar, 20 μm. B. Arp3 recruitment to the contractile ring is not affected in N-WASP null-cells. HeLa in cytokinesis transfected with siRNA as previously described were treated for indirect Alexa 555 localisation of Arp3 (a, e and i) and Alexa 350 localisation of β-tubulin (c, g and k) using specific antibodies. F-actin was stained with FITC-coupled phalloidin (b, f and j). Merged images (d, h and l) are presented. Exposure times in Alexa 555 and FITC channels were fixed as previously described. Arp3 recruitment to the contractile ring is identical between control (a, arrowhead) and N-WASP (b, arrowhead) null-cells but is greatly reduced in Arp3 null-cells (i, arrowhead). Bar, 10 μm. C. Time course depletion of N-WASP, Arp3 and ECT2 proteins by RNA interference. HeLa cells were transfected with indicated siRNA and were harvested 24, 48 and 72 hours after transfection. Proteins levels were determined by immunoblot analysis using specific antibodies. The maximum of N-WASP knockdown is achieved after 48 hours transfection, whereas 72 hours are required for Arp3 and only 24 hours for ECT2.
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Bompard et al. BMC Cell Biology 2008 9:42 doi:10.1186/1471-2121-9-42