In vitro guanine nucleotide exchange assay

All purified proteins were quantified for their concentrations and subjected to Zeba Spin Desalting Column (molecular weight cut-off 7 kDa) (Thermo Fisher Scientific) to change their buffers to HK buffer containing 50 mM HEPES pH 7.5, 120 mM KCl and 1 mM DTT. Guanine nucleotide exchange reactions were assembled in a black 96-well plate (Greiner Bio-One) in 150 µl HK buffer containing additional 1 mM MgCl₂, 1 µM Mant-GMPPNP (Thermo Fisher Scientific), 1 µM His-Δ14Arl5b and 0.7 µM EDTA, Ragulator or Ragulator subcomplex. The change of fluorescence was monitored in Cytation 5 (BioTek) at 26 °C with excitation at 360 nm and emission at 440 nm. The fluorescence data were collected every 50 s and subsequently subjected to single exponential curve-fitting (y = y₀+ A * exp(−(x−x₀)/τ)) in OriginPro2015 (Origin Lab). All fitted data had adjusted-R² ≥ 0.95.