Figure 7.
Activation of EB3 reverse signaling leads to increased Rac and Cdc42 GTP levels. a and b, Expression of a dominant-negative Rac1, mCherry-Rac1-N17, blocks axon pruning initiated by co-culture of primary hippocampal neurons with CellTracker labeled Cos-1 cells (blue) for 48 h in live cell assays (n = 13–27 transfected neurons per group, *P < 0.0001). Mean ± s.e.m. Scale bars: 20 μm. c, GTP-bound Rac1 and Cdc42 were precipitated by GST-PDB fusion protein pull downs from EB3-WT expressing NG108 cells following a time course of EphB2-Fc stimulation of reverse signaling. The immunoblots show a biphasic activation of Rac1 and Cdc42 in response to EphB2-Fc. Arrow indicates increased EB3 tyrosine phosphorylation. d, GTP-bound Rac1 and Cdc42 were precipitated by GST-PDB fusion protein pull downs from EB3-WT and EB3-3F expressing NG108 cells following 30 min EphB2-Fc stimulation of reverse signaling. The immunoblots show that Rac1 and Cdc42 are activated by EphB2-Fc stimulation in EB3-WT cells but not in EB3-3F cells.