Detection of Arf1-GTP in cells using a GST-GGA3 pull-down assay

Activation endogenous Arf1 by transfection of mRNA or by infection was monitored using GST-GGA3 pull-down assay described in [41] with minor changes. Cells were plated at a density of 3 X 10⁶ cells in 100 mm dish 24 h prior to transfection or infection. 3CD (WT or mutant) mRNA (15.5 μg) was transfected into HeLa cells using TransIT-mRNA transfection kit (Mirus). Four hours post-transfection or post-infection cells were lysed at 4°C in 0.5 mL of lysis buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 10 mM MgCl₂, 0.1% SDS, 0.5% sodium deoxycholate, 1% triton X-100, 5% glycerol with 0.1 mM PMSF and 1 μg/mL each of pepstatin and leupeptin).

Lysates were incubated on ice for 5 min in presence of 50 μL of CL-4B Sepharose beads (per sample) and then centrifuged for 15 min at 16,000 X g and 4°C. The clarified lysate (500 μg) was incubated with 40 μg of GST-GGA3 bound to Glutathione Sepharose beads for 1 h on a rotating shaker at 4°C. The beads were then washed three times with 1 mL cold lysis buffer followed by a final wash in 1 mL cold PBS (1X). Liquid was removed beads by quick inversion followed by and a 30 s spin at 6,000 X g at 4°C and removal of residual buffer using a pipetman. Bound proteins were eluted from beads by adding 30 μL of 2X SDS-PAGE sample buffer and incubating at 65°C for 10 min. Beads were pelleted by centrifugation at 6,000 X g for 2 min at room temperature. Twenty μL of the supernatant was resolved by SDS polyacrylamide gel electrophoresis. Endogenous, activated Arf1 was detected using anti-Arf1 antibody. For Total Arf1 was detected using the same antibody, but the sample was prepared from 10 μg of the clarified lysate. Alkaline phosphatase activity associated with the secondary antibody was detected using the ECF reagent (GE Healthcare) and quantified by imaging the fluorescence signal using the Syngene G-box.