Localization of brain CDC42 (CDC42b) and ubiquitous CDC42 (CDC42u) in migrating astrocytes. (A) Confocal section images of migrating astrocytes 3–4 h following microinjection of GFP-tagged CDC42u or CDC42b constructs and 5 h after wounding. The right panels show higher magnification of the corresponding boxed area and highlight the different localization of the two isoforms (see corresponding Videos 2 and 3). (B) Confocal stack images of GFP-CDC42u- and mCherry-CDC42b-expressing astrocytes 5 h after wounding stained with anti-GM130 (cis-Golgi marker). Right panel, quantification of the colocalization of each CDC42 construct with GM130. (C) Confocal stack images of GFP-CDC42u and mCherry-CDC42b expressing astrocytes 5 h after wounding stained with anti-EEA1 (early endosomes marker). Right panel, quantification of the colocalization of each CDC42 construct with EEA1. (D) Spinning disk section images showing localization of GFP-tagged non-lipid-modified CDC42u (u[SVLL]), non-prenylated brain CDC42 (b[SCIF]), non-palmitoylatable CDC42b (bCSIF), and/or non-lipid modified CDC42b (bSSIF) in migrating astrocytes 5 h after wounding. Right panel, showing the quantification of nucleus/cytoplasmic ratios for each lipid mutant. All graphs show the values and means ± SEM of three independent experiments, and at least 30 cells were analyzed per condition. All P values were calculated using two-sided unpaired Student’s t test. Scale bars: 10 µm.