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. Author manuscript; available in PMC: 2010 Nov 1.
Published in final edited form as: Neurobiol Dis. 2009 Aug 20;36(2):374–383. doi: 10.1016/j.nbd.2009.08.003

Figure 2. Impaired extraction of Rab11 by RabGDI contributes to elevation of Rab11 on membranes in HD.

Figure 2

A, Post-debris supernatants and total membranes prepared from striata of young (10 – 14 weeks old) WT and HD mice as described in Methods were analyzed by SDS-PAGE and Western blot analysis with indicated antibodies. Shown are a series of blot analyses. B, Quantitative data for A. Films were scanned and the band density was measured using NIH ImageJ. The density of calnexin was used for normalization of loading variations. Data are represented as ratio of Rab11 on HD striatal membranes to Rab11 on WT striatal membranes (n=6 mice for each of WT and HD, Mean±SD, Student t-test: * p<0.01). C, RabGDI-mediated extraction of Rab11 was performed as described in Methods. In this assay we used 20μg HD membranes and 60μg WT membranes to keep the same level of Rab11GDP to be retrieved by RabGDI. The extracted Rab11 (released) in complex with GST-RabGDI on glutathione and the un-extracted Rab11 (unreleased) in the supernatant was precipitated with chloroform/methanol and was boiled in SDS-PAGE sample buffer. Samples were analyzed by SDS-PAGE and Western blot with anti-Rab11 and anti-GDI antibodies. Shown are blot analyses from one of the three experiments. D, Densitometry data obtained in C were used to calculate the percentage of extracted Rab11 by GST-RabGDI by dividing the extracted amount from the total input (released + unreleased). Average percentage of released Rab11 by GST-RabGDI was plotted (n=3, Mean±SD, Student t-test: * p<0.001).