Localization of Pyk2 in unstimulated and fMLP-stimulated adherent dHL-60 cells. dHL-60 cells (2 × 105) were added to fibrinogen-coated 8-well chamber slides with or without 1 μM fMLP for 30 min. Pyk2 localization in unstimulated (A) or fMLP-stimulated cells (B-C) was detected by anti-phospho Pyk2 antibody. Phospho-Pyk2 was localized adjacent to cell membrane as a round circle in non-stimulated control cells, while it concentrates on pseudopods and uropods of polarizing cells (B) in a punctate staining after fMLP stimulation. The lower panel of figure A and B were transmission images. C) Colocalization of Pyk2 and F-actin in dHL-60 at lamellipodia. dHL-60 cells (2 × 105) were added to fibrinogen-coated 8-well chamber slides with 1 μM fMLP for 30 min. Pyk2 localization in dHL-60 cells was detected by anti-phospho Pyk2 antibody (Green, upper left). F-actin was detected by Alexa Fluor 647 phalloidin staining (Red, upper right). The transmission image was shown in lower left, and the colocalization of Pyk2 and F-actin was shown in lower right in yellow. Representative images were shown from four experiments. Arrow points the colocalization in lamellipodia.