Figure 2.

Detection of ARNO-mediated nucleotide exchange using the GST-GGA^GAT binding assay. (a,b) One µM GDP preloaded ^NΔ17HsArf1 (a) or ^NΔ17PfArf1 (b) was incubated with 0.2 µM ARNO^Sec7 and 50 µM GTP for 30 min at 37 °C, added to Ni-NTA coated 96-well plates and incubated for a further 30 min at 4 °C. GST-GGA3^GAT was added to 1 µM and incubation continued at 4 °C for 60 min, followed by washing, incubation with GST substrate and absorbance readings at 340 nm. Control incubations contained the respective GDP preloaded Arf1 proteins alone, GDP preloaded Arf1 incubated with ARNO^Sec7 in the absence of GTP and GDP preloaded Arf1 incubated with GTP in the absence of ARNO^Sec7. (c–f) ARNO^Sec7 nucleotide exchange reactions were repeated with GDP preloaded ^NΔ17HsArf1 and ^NΔ17PfArf1in the presence of 50 µM SecinH3 (c,d) Brefeldin A (BFA) or Golgicide A (GA) (e,f), followed by the GST-GGA3^GAT binding assay. Control reactions consisted of the GDP preloaded Arf1 proteins incubated with GTP in the absence of ARNO^Sec7 and inhibitors. Mean Abs₃₄₀ values obtained from empty Ni-NTA plate wells incubated with GST-GGA3^GAT were subtracted from all other readings. Incubations were carried out in triplicate wells and Abs₃₄₀ shown as mean ± standard deviation. P-values were derived from two-tailed t-tests.