MKL1-deficient neutrophils are defective in actin polymerization and have reduced actin levels. Actin polymerization was examined in suspension after stimulation with fMLF (10 nM) or C5a (10 nM) (mean ± range, n = 2 of the index patient) (A) and by confocal analysis with staining for F-actin (magenta = F-actin [phalloidin], cyan = DNA [Hoechst]) upon a 10-minute stimulation with fMLF (B). Images were acquired with a Leica TCS SP8 confocal microscope. Scale bars, 10 µm. (C) Cytospins of isolated neutrophils from control and index patient samples (original magnification ×400; May-Grünwald-Giemsa stain). (D) Reduced actin levels in MKL1-deficient neutrophils were detected using western blotting with anti-actin. GAPDH was used as loading control (n = 2 of the index case). (E) Quantification of actin levels in panel D. (F) Representative immuno-electron microscopy images stained for anti-actin (black dots). The pseudopodia were more bluntly shaped compared with control neutrophils (lower panels). Images were acquired with a Tecnai 12 G2 transmission electron microscope. Scale bars, 1 µm. (G) Quantification of gold-labeled particles in immuno-electron microscopy images stained for anti-actin (n = 1 of the index patient and control; 4 images were analyzed per donor). C, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; P, patient.