Soluble fractions of cell lysate containing all His-tagged APPL1 (5–419) variants were analyzed by SDS–PAGE to confirm their comparable expression level and solubility.
The GSH resin carrying nucleotide-loaded GST−Rab fusion protein was incubated with ∼50 μl cell lysate (∼200 μg APPL1 variant, 50 kDa) at 22°C for 30 min, then washed three times with 200 μl of (1 × PBS, 2 mM DTT, and 4 mM MgCl2) and resuspended in 20 μl of 2 × reducing SDS sample buffer.
The sample was subjected to SDS–PAGE analysis, visualized with Coomassie blue stain.