Fig. 4

Thy-1-induced astrocyte migration requires PAR-3-dependent Tiam1 activation. a PAR-3 silenced DI TNC1 astrocytes were stimulated with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL) for 60 min. Cells were fixed, and active Rac1 GEF was detected by immnunofluorescence using GST-Rac1G15A and anti-GST (purple) and Phalloidin (green). The white rectangle indicates the zoomed area. The color map indicates levels of Rac1 GEF activation, going from 0 (blue) to 256 (white). Scale bar = 10 μm. b DI TNC1 cells transfected with siRNA control (siCTRL), siRNA Tiam1 (siTiam1), or dominant negative Tiam1 (Tiam1-DN). The Western blot shows levels of Tiam1 in siRNA-transfected cells. Migration was evaluated using the wound-healing assay, and cells were stimulated as in (a) for 24 h. Images show wound closure at time zero (green) and after 24 h (red). c DI TNC1 cells stimulated or not (NS) with endogenous Thy-1 from fixed CAD cells for different periods of time. Active Tiam1 was affinity-precipitated using GST-Rac1G15A coupled to GSH-agarose beads and immunoblotted for Tiam1. Numbers in the graphs represent the average fold-increase of Tiam1 activity normalized to total Tiam-1. All graphs show the mean ± s.e.m. of n = 3. Significant differences compared to control conditions are indicated as *P < 0.05