The Journal of Neuroscience, February 9, 2005, ():
A Truncated Tropo-Myosine-Related Kinase B Receptor, T1, Regulates Glial Cell Morphology via Rho GDP Dissociation Inhibitor 1
J. Neurosci. Ohira et al.
25: 1343
Supplemental data
Files in this Data Supplement:
- supplementary material
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Supplemental Figure 1. Production of GST fusion proteins of T1-ICD and Rho GDI1. (A) GST-T1-ICD was produced in E. coli cells. The cell lysates were incubated with Glutathione-Sepharose 4B, and an aliquot of Glutathione-Sepharose 4B slurry was boiled in SDS sample buffer. The arrow and arrowheads indicate GST (26 kDa) and GST-T1-ICD (27 kDa), respectively. (B) GST-Rho GDI1 was expressed in E. coli cells. The cell lysates were incubated with Glutathione-Sepharose 4B, and an aliquot of Glutathione-Sepharose 4B slurry was boiled in SDS sample buffer and subjected to Western blot analysis (the second and third lanes from the left). The GST-Rho GDI1 bound to Glutathione-Sepharose 4B was cleaved by Factor Xa. Then, we confirmed the cleavage of GST-Rho GDI1 by Western blot analysis (see the second to the fifth lanes from the left). The molecular weight of GST-Rho GDI1 before cleavage is about 54 kDa, whereas that of Rho GDI1 is about 28 kDa. The arrowheads and arrow indicate GST-Rho GDI1 (54 kDa) and Rho GDI1 (28 kDa), respectively. The resulting GST-T1-ICD and Rho GDI1 were subjected to the following experiment (Fig. 2A).
- supplemental material
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Supplemental Figure 2. Control of Rho GTPase pull-down assays. The extracts from 30 DIV astrocytes were incubated with GTPãS and GDP at 30 oC for 30 min. After precipitation with Glutathione-Sepharose 4B, the precipitates were analyzed using Western blotting. The precipitates and total proteins (5 µg for RhoA; 100 µg for Cdc42 and Rac1) were loaded on 15% gel, and detected with specific antibodies. In our experimental system, we were able to detect the activities of the Rho GTPases.
