Nupage acrylamide gel

In vitro radioactive assays were performed by incubating 100 ng recombinant ATG4B diluted in assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 5 mM DTT, 20 µM cold ATP and 0.16 µM ATP [γ-32P] PerkinElmer NEG502A100UC) in the presence of 10 ng recombinant ULK1 (catalytic domain) or 3 µL of active recombinant ULK1 (Sigma-Aldrich, SRP5096) at 30 °C for the indicated times. The reaction was stopped by adding 5× sample buffer (250 mM Tris-Cl pH 6.8, 10% sodium dodecyl sulphate (SDS), 50% glycerol, 25% β-mercaptoethanol or 0.5 M DTT and 0.05% bromophenol blue) and boiling for 5 min. Samples were loaded on NUPAGE Acrylamide gel (Invitrogen, NP0321BOX). Gels were stained with InstantBlue Protein Stain (Expedeon, ISB1L) before drying on filter paper and measuring incorporated radioactivity by exposing on photographic film (Bio-Rad).

In vitro radioactive assays were performed by incubating 100 ng recombinant ATG4B diluted in assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 5 mM DTT, 20 μM cold ATP, 0.16 μM ATP [γ-³²P] Perkin-Elmer NEG502A100UC) in the presence of recombinant AKT2 (Sigma-Millipore) at 30°C for the indicated time. The reaction was stopped by adding 5X SDS Loading Buffer and boiling for 5 min. Samples were loaded on NUPAGE Acrylamide gel (Invitrogen, NP0321BOX). Gels were stained with InstantBlue Protein Stain (Expedeon, ISB1L) before drying on filter paper and measuring incorporated radioactivity by exposing on photographic film (Bio-Rad).