Mix 1 μM ATG3 WT or mutant, 1 μM ATG7, 500 nM WIPI2d, 1 μM ATG12–ATG5-ATG16L1, 5μM LC3B

and 0.5 mg/mL extruded liposomes in reaction buffer (25mM HEPES pH 7.4, 150 mM NaCl, 1 mM TCEP).

Incubate at room temperature for 20 minutes.

Quench with 4X LDS loading buffer. Boil samples for 10 minutes at 60ºC. Load and run SDS PAGE gel

Visualize protein bands with coomassie blue