Fig. 3. Direct interaction between RILP and Rab7 in vitro. (A) GST and GST-tagged Rab7T22N and Rab7Q67L were expressed in BL21 E.coli cells, purified and immobilized on a glutathione resin. They were incubated with 100 µM GDP (Rab7T22N) or GTP (Rab7Q67L) and then with extracts of HeLa cells. Samples were then loaded on an SDS–polyacrylamide gel and subjected to western blot analysis using anti-RILP polyclonal antibodies. (B) One (lanes 1 and 3) or 2 (lanes 2 and 4) µg of GST-tagged RILP expressed in BL21 E.coli cells were loaded onto an SDS–polyacrylamide gel and transferred to nitrocellulose. The blot was then renatured as described in Materials and methods. A 10 µg aliquot of GST–Rab9 (lanes 1 and 2) or GST–Rab7 (lanes 3 and 4) loaded with [α-³²P]GTP was applied to the renatured blot.