GGA3-mediated Pulldown of ARF Proteins

Method described previously (Nacke et al., 2021 (link)); briefly, PC3 cells were incubated on 120-mm plates for 48 h at 37°C, 5% CO₂. Cells were then lysed on ice in pulldown-lysis buffer (50 mM Tris, 100 mM NaCl, 2 mM MgCl₂, 0.1% SDS, 0.5% Na-deoxycholate, 1% Triton X-100, 10% glycerol). Lysates were syringed 5× using a 25-27G needle and centrifuged at 4°C 14,000 g for 1 min. Spin columns were equilibrated with 50 μl of Glutathione Agarose resin and washed with pulldown-column wash buffer (1:1 pulldown-lysis buffer and 1×TBS). 80 μg of GST-GGA3-GAT recombinant fusion protein was immobilized on the agarose resin by incubation at 4°C with gentle rocking. After 1 h, 1 mg of each lysate was added onto spin columns and incubated again at 4°C for 2 h with rocking. Pulldown wash buffer (50 mM Tris, 100 mM NaCl, 2 mM MgCl₂, 1% NP-40, 10% glycerol) was used to wash unbound proteins off the column. 60 μl of pulldown-elution buffer (10 mM Glutathione in 1×TBS) was added to each spin column and incubated for 5 min at room temperature. Eluted protein was collected at 1,250 g for 1 min and samples prepared for SDS-PAGE and immunoblotting as described above. n = 3 experimental replicates for ARF3 Chimeras and n = 5 for PSD KD. Data is presented as GGA3 binding normalized to ARF3 or ARF6 levels for each experiment. Values are mean ± SEM.