Plan apochromat 1.4 na objective

The reactions were set up in an eight-well observation chamber (Lab Tek) at room temperature. The chamber was coated with 5 mg/ml β casein for 30 min and washed three times with reaction buffer (20 mM HEPES at pH 8.0, 190 mM NaCl and 1 mM TCEP). A final concentration of 10 µM GST-4xUb, 1 µM NDP52, and 100 nM GFP-FIP200 or GFP-ULK1 complex was used for all reactions unless otherwise specified. 15–20 µL GUVs were added to initiate the reaction in a final volume of 150 µL. After 5 min incubation, during which we picked random views for imaging, time-lapse images were acquired in multitracking mode on a Nikon A1 confocal microscope with a 63 × Plan Apochromat 1.4 NA objective. Three biological replicates were performed for each experimental condition. Identical laser power and gain settings were used during the course of all conditions.

A mixture of 1 µM GST-NDP52 and 100 nM GFP-ULK1 complex was incubated with 10 µL Glutathione Sepharose beads (GE Healthcare) in a reaction buffer containing 20 mM HEPES at pH 8.0, 200 mM NaCl and 1 mM TCEP. After incubation at room temperature for 30 min, the beads were washed three times, suspended in 120 µL reaction buffer, and then transferred to the observation chamber for imaging. Images were acquired on a Nikon A1 confocal microscope with a 63 × Plan Apochromat 1.4 NA objective. Three biological replicates were performed for each experimental condition.