The Journal of Neuroscience, April 27, 2005, ():
Involvement of a Rac Activator, P-Rex1, in Neurotrophin-Derived Signaling and Neuronal Migration
J. Neurosci. Yoshizawa et al.
25: 4406
Supplemental data
Files in this Data Supplement:
- supplemental material
-
Fig. S1
GTP-bound Rac1 in PC12 cells transfected with indicated constructs used in the trans-well migration assay, with or without NGF stimuli. (a) PC12 cells were transfected with pEGFP-N2 (lanes 1, 2), pEGFP-DDH-Pex1 (lanes 3, 4), pcDNA3-Myc-N17Rac1 (lanes 5, 6), pEGFP-FL-P-Rex1 plus pcDNA3 (lanes 7, 8), and pEGFP-FL-P-Rex1 plus pcDNA3-Myc-N17Rac1 (lanes 9, 10). After transfection, cells were incubated in the culture medium at 37°C for 24 h, serum starved for 16 h, and then treated with or without 50 ng/ml NGF for 3 min. Left upper panel shows GTP-bound Rac1 (arrowhead). Left lower panel shows total Rac1. Right panels depict proteins derived from transfected constructs. (b) Introduction of R-Pex1 siRNA. pEGFP-N2 was cotransfected with control Luciferase GL2 siRNA or P-Rex1 siRNA into PC 12 cells using Cell Line NucleofectorTM Kit V (Amaxa), and incubated in normal culture conditions for 4 days. After treatment of 50 ng/ml NGF for 3 min, cells were lysed and Rac pull-down GTPase assays were performed.
Fig. S2
Effects of DDH-P-Rex1 on NGF-induced Rac1 activities of PC12 cells. pEGFP-N2 was cotransfected with pcDNA3 (lanes 1, 2) or pMyc-DDH-P-Rex1 (lanes 3, 4) into PC12 cells by using Cell Line NucleofectorTM Kit V (Amaxa). After an administration of 50 ng/ml NGF for 3 min, cells were lysed and Rac pull-down GTPase assays were performed. Left upper panel indicates GTP-bound Rac1. Left lower panel highlights total Rac1. Right panel depicts proteins derived from transfected constructs. Arrowhead indicates endogenous GDP-bound Rac1 protein.
