Plectin knockdown phenocopies IF depletion. (A) Immunofluorescence images of migrating astrocytes stained for nuclei (cyan), paxillin (green), and plectin (magenta). (B) Western blot analysis using indicated antibodies of total astrocyte lysates (left) and proteins immunoprecipitated with antibodies against plectin, talin, or vinculin and control antibodies (IgG Ms) before and 4 h after wounding. Input lysate corresponds to 2% of the total lysate used for the IP, with a higher exposition shown for plectin and talin bands. (C) Immunofluorescence images of migrating astrocytes stained for nuclei (cyan) and actin (phalloidin, gray) compared with si ctl (Fig. 2 A). The white dotted line represents the position of the wound. The quantification shows the percentage of cells that present ITAs. (D) Immunofluorescence images of migrating astrocytes stained for the AJ marker α-E-catenin (gray) and nuclei (cyan). The white dotted line represents the position of the wound. (E) Quantification of nuclear tracking of migrating astrocytes after 24 h of migration. The graphs show cell velocity, directionality, and persistence of migration of astrocytes nucleofected with si ctl or si plectin. (F) Immunofluorescence images of centrosome orientation in plectin-depleted astrocytes stained for nuclei (cyan) and centrin (red) compared with si ctl (Fig. S1 F). White arrowheads were scored as polarized centrosomes, and yellow ones were scored as nonpolarized centrosomes. The graphs show the percentage of cells with the centrosome located in the wound-facing quadrant in front of the nucleus. Data are from n = 3 independent experiments. The sample size for each repeat: si ctl 262, 247, 150 and si plectin 384, 245, 155 for C; si ctl 40, 50, 39 and si plectin 50, 50, 49 for E; si ctl 92, 222, 83 and si plectin 101, 210, 123 for F. ***, P < 0.001. Histograms show mean ± SEM. Bar, 10 µm.