Fig. 2. RIN3 and RIN2 act as GEFs and stabilizers for Rab5b. (A) Flag-tagged RIN3, RIN2 (left) and prenylated Rab5b (right) purified from baculovirus-infected Sf9 cells were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. (B) The purified Rab5b (18 nM) that had been preloaded with 5 µM [³H]GDP was subjected to the nucleotide exchange assay in the presence of 300 nM RIN3 (triangles), RIN2 (squares) and Flag-peptide alone (circles). At the indicated times, an aliquot was removed from the reaction mixture, quenched and filtered through a nitrocellulose membrane. The membranes were subjected to liquid scintillation counting. The proportions of [³H]GDP retained in the membranes are presented as a function of the incubation times. (C) The purified Rab5b (18 nM) was incubated at 30°C with 1 µM [³⁵S]GTPS in the presence of 300 nM RIN3 (triangles), RIN2 (squares) and Flag-peptide alone (circles). The amounts of [³⁵S]GTPS bound to Rab5b are presented as a function of the incubation times. [³⁵S]GTPS-binding activity was not detected in the fraction of RIN3 or RIN2 (data not shown). (D) Lysate was prepared from COS7 cells that had been transfected with Flag-RIN3 and immunoprecipitated with the anti-Flag antibody-conjugated resin. The resin was washed and incubated with or without GDP- or GTPS-bound Rab5b. Proteins bound to the resin were separated by SDS-PAGE and immunoblotted (IB) with the anti-Flag (top) and anti-Rab5 (bottom) antibodies.