Fig. 3. The VPS9 domain of Varp catalyzes Rab21 guanine nucleotide exchange reaction in vitro. (A) A schematic diagram of the full-length Varp and VarpN protein. VPS9, vacuolar protein sorting 9 domain; ANK, ankyrin repeat. (B) Flag-tagged Varp and VarpN proteins purified from transfected HEK293T cells were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. (C) [³H]GDP-loaded His₆-Rab5a was incubated with the purified Flag-tagged Varp, VarpN and Rabex5. The percentage of [³H]GDP remained bound to Rab5a after 60 minutes is presented. Each value represents the mean and standard deviation of at least two independent assays. (D) His₆-Rab5a was incubated with [-³⁵S]GTP at 30°C in the presence of Flag-tagged Varp, VarpN or Rabex5 for 60 minutes. Counts of the bound [-³⁵S]GTP on Rab5a are presented. Each value represents the mean and standard deviation of at least two independent assays. (E) A total of six Rab GTPases, including Rab4, Rab5a, Rab7, Rab15, Rab21 and Rab22, were subjected to the in vitro [-³⁵S]GTP-binding assay. Each GST-fused Rab GTPase was incubated with [-³⁵S]GTP at 30°C in the presence or absence of Flag-tagged VarpN for 60 minutes. Each value represents the mean and standard deviation of at least two independent assays. Empty, no Rab protein.