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. 2006 Sep;74(9):5362–5373. doi: 10.1128/IAI.00539-06

FIG. 5.

FIG. 5.

GST-Rab4A interacts with GFP-CT229. GST-Rab4A and GST were expressed in E. coli and purified by affinity chromatography using glutathione agarose beads (data not shown). (A) Glutathione-bound GST-Rab4A was loaded with GTPγS and incubated overnight with protein extracts from HeLa cells expressing GFP or GFP-CT229. Beads were washed, and bound protein was eluted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis was performed using anti-GFP. Lane 1, total cellular lysate from GFP-expressing HeLa cells. Lane 2, total cellular lysate from GFP-CT229-expressing HeLa cells. GST-Rab4-GTPγS (lane 6) but not GST (lane 5) interacted with GFP-CT229. Neither GST-Rab4-GTPγS (lane 4) nor GST (lane 3) pulled down GFP. The thin arrow indicates GFP-CT229. The thick arrow indicates GFP. (B) Glutathione-bound GST-Rab4A was loaded with GTPγS or GDP and incubated overnight with protein extracts from HeLa cells expressing GFP-CT229. Beads were washed, and bound proteins were eluted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis was performed using anti-CT229. Lane 1, total cellular extract of GFP-CT229-expressing HeLa cells; lane 2, total cellular extract of GFP-expressing HeLa cells. GST-Rab4-GTPγS (lane 3) but not GST-Rab4-GDP (lane 4) interacted specifically with GFP-CT229. The arrow indicates GFP-CT229. Lane 5, total cellular extract of GST-expressing HeLa cells. The asterisk indicates cross-reacting protein present in HeLa cells.