Expression of Syk mutants or the down-regulation of Syk by RNAi technique compromised the enrichment of Syk and Vav at the leading edge and resulted in the formation of multiple lamellipodia. Confocal microscopy images of human Syk detected by Syk-EGFP epifluorescence (green) or a mouse anti–human Syk mAb and a secondary Alexa 546–conjugated anti–mouse IgG Ab (red). F-actin was assessed using Alexa 488–conjugated phalloidin (green). Vav was detected using a rabbit anti-Vav Ab and a secondary Alexa 546–conjugated anti–rabbit IgG Ab (red). (A) Schematics of the Syk constructs. (B) Upon induction of adhesion by fMLP (100 nM) on immobilized fibrinogen, Syk and Vav were enriched at the leading edge (arrow) of EGFP-Syk transfectants. The merged images demonstrate colocalization (yellow). In contrast, EGFP-Syk K402R and EGFP-Syk Y348F transfectants showed no enrichment of Syk and Vav at the leading edge (arrowheads). (C) Western blot analysis of a Syk-siRNA clone (siRNA) and wild-type HL-60 cells (control) using an anti-Syk mAb and an anti–β-actin antibody for control. Numbers indicate relative optical density (OD) of Syk protein expression. (D) Upon induction of adhesion by fMLP (100 nM) on immobilized fibrinogen, the down-regulation of Syk resulted in the formation of multiple lamellipodia (arrowheads) and abolished the redistribution of Syk and Vav at the leading edge as seen in control cells. Transmission images show the morphology of the corresponding cell (transmission). Scale bar equals 10 μm; n = 3.