Two-dimensional differential in-gel electrophoresis of neutrophil lysates. (A) Lysates of normal and patient I.2.2 neutrophils were differentially labeled (with Cy3, green, and Cy5, red, respectively) and mixed together, and the proteins were resolved by 2D gel electrophoresis (isoelectric focusing horizontally, and SDS-PAGE vertically). The protein spots on the resolved proteins gels were imaged for the red and green dye fluorescence, respectively, and then the ratios of the images were determined. Green spots indicate increased protein expression in the lysate of neutrophils from the normal subject compared with the lysate from neutrophils of patient I.2.2; similarly, red spots indicate increased protein expression in the lysate of neutrophils from patient I.2.2. Convergence of spots resulting in yellow indicated equivalent protein expression in the lysates of neutrophils from both subjects. The spots that were differentially expressed are indicated by circles and identified by numbers. The identity of these differentially expressed spots was excised and determined by mass spectrometry. The inset is an enlargement of the region where Aip1 migrated. (B) Same analysis for normal compared with patient I.2.3. (C) Relative changes in the mean (normal vs patient) expression of proteins in neutrophil lysates. The number in each bar refers to the specific spot on the 2D gel. Note that both cofilin (*) and AIP1 (arrows) are represented in multiple spots. (D) Protein expression in neutrophil lysates from 3 normal subjects, patients I.2.2 and I.2.3, and their unaffected heterozygous sibling, I.2.1, and patient III.2.2 of family III. The immunoblots were probed with antibodies to Aip1, cofilin, phospho-cofilin Ser(3), and actin as indicated.