Figure 1. Rab29 binds nucleotide weakly... | Rockefeller University Press

Figure 1.

$Figure 1. Rab29 binds nucleotide weakly and does not bind GDI in the cytosol. (A–D) Purified His-Rab5, His-Sumo Rab29, or His-Sumo Rab29 D63A was loaded with MANT-GDP, and the release of MANT-GDP fluorescence was followed after addition of an excess of unlabeled GDP. Shown are examples of experiments performed at least three times. A and C are normalized to time zero; B and D are unnormalized, and a longer time course is shown. (E) The curves from B and D were fitted to a nonlinear regression one-phase decay function. Times of 50% dissociation from three experiments performed in duplicate are shown. Error bars represent SEM. (F) Cytosol collected from 293T cells transfected with either Myc-Rab8A or Myc-Rab29 were applied onto an Sephacryl S100 size exclusion column and fractions immunoblotted using antibodies to GDI, Myc, or endogenous Rab9. (G) HEK392T cells were transfected with GFP-Rab10 or GFP-Rab29. Cell lysates were incubated with biotin-labeled geranyl pyrophosphate for 4 h at RT. GFP-Rabs were immunoprecipitated using beads to which GFP-binding nanobody was attached and immunoblotted for incorporated biotin geranyl pyrophosphate using streptavidin-IR800 and chicken anti-GFP antibody for total Rab protein. Shown is an example of an experiment performed three times. The molecular mass is shown at left in kilodaltons in this and all subsequent figures.$

Rab29 binds nucleotide weakly and does not bind GDI in the cytosol. (A–D) Purified His-Rab5, His-Sumo Rab29, or His-Sumo Rab29 D63A was loaded with MANT-GDP, and the release of MANT-GDP fluorescence was followed after addition of an excess of unlabeled GDP. Shown are examples of experiments performed at least three times. A and C are normalized to time zero; B and D are unnormalized, and a longer time course is shown. (E) The curves from B and D were fitted to a nonlinear regression one-phase decay function. Times of 50% dissociation from three experiments performed in duplicate are shown. Error bars represent SEM. (F) Cytosol collected from 293T cells transfected with either Myc-Rab8A or Myc-Rab29 were applied onto an Sephacryl S100 size exclusion column and fractions immunoblotted using antibodies to GDI, Myc, or endogenous Rab9. (G) HEK392T cells were transfected with GFP-Rab10 or GFP-Rab29. Cell lysates were incubated with biotin-labeled geranyl pyrophosphate for 4 h at RT. GFP-Rabs were immunoprecipitated using beads to which GFP-binding nanobody was attached and immunoblotted for incorporated biotin geranyl pyrophosphate using streptavidin-IR800 and chicken anti-GFP antibody for total Rab protein. Shown is an example of an experiment performed three times. The molecular mass is shown at left in kilodaltons in this and all subsequent figures.