Tris base

Lysates and eluates from Immunoprecipitations were run on 10% acrylamide gels for SDS–PAGE, freshly prepared and used the same day: 10% Protogel (30% w/v acrylamide, 0.8% bis‐acrylamide (37.5:1 solution, National diagnostics, Atlanta, USA), 380 mM Tris–HCl pH 8.8, 0.1% w/v SDS (Applichem, Darmstadt, Germany), 0.06% v/v TEMED (Applichem), 0.06% w/v APS (Applichem) for the running gel and 5% Protogel, 165 mM Tris–HCl pH 6.8, 0.1% w/v SDS, 0.08% v/v TEMED, 0.04% w/v APS for the stacking gel. Running buffer for SDS–PAGE was 190 mM glycine (Applichem), 25 mM Tris‐base (Applichem), 0.5% SDS. To facilitate Atg18 migration and avoid formation of aggregates, samples were reduced and denatured at 90°C using NuPAGE buffer (Thermo Fisher) containing LDS instead of SDS and supplemented with 100 mM DTT. Gels were blotted on 0.45 µm nitrocellulose membrane (Amersham) overnight at a constant current of 200 mA using a Trans‐Blot® Cell (Bio‐Rad, USA). Membranes were decorated using anti‐mCherry‐1C51 (Abcam), anti‐HA.11‐16B12 (BioLegend), anti‐G6PDH (Sigma‐Aldrich), anti‐Tubulin (clone B5‐1‐2, Sigma‐Aldrich), and anti‐WIPI1 (C‐terminal epitope, Sigma‐Aldrich).