Protogel

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).

Purified DNA was assessed using 1X TBE 8% polyacrylamide gels prepared for a Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad) using ProtoGel (National Diagnostics) acrylamide and run at 100 V. Native PAGE gels were stained with 1X SYBR Gold in 1X TBE for 10 min and scanned on a Sapphire Biomolecular Imager using the 488-nm/518BP22 setting.

Iso-electrofocusing gels were prepared as detailed (3.15 g of Urea, 2.1 mL of 30% acrylamide/bisacrylamide (Protogel, National Diagnostics, Nottingham, UK), 0.35 mL of pH = 2.5 to 5 ampholyte (GE), 0.175 mL of pH = 3 to 10 ampholyte (GE), 4.725 mL of ultrapure water, 5.25 µL of TEMED, 105 µL of 100 g/L APS. As an anolyte buffer 0.1 M glutamic acid, 0.5 M phosphoric acid was used and as a catholyte buffer 0.89% beta-alanine. The gel was pre-run for 60 min at a constant power of 5 W, with maximum amperage of 50 mA. The focusing was carried out for further 180 min at a constant power of 7 W, with maximum amperage of 50 mA. After separation, the gel was placed in the in equilibration buffer (125 mM Tris pH = 6.8, 5% 2-mercaptoethanol, 1% SDS) for 20 min at 4 °C and subsequently into transfer buffer (25 mM Tris, 192 mM Glycine) for 20 min at 4 °C. Separated EPO isoforms were then transferred to PVDF membrane and probed with primary anti-EPO antibody a 1:1000 dilution (rabbit anti-EPO R&D) for 1 h at room temperature followed by incubation with anti-rabbit (HRP-conjugate) secondary antibody diluted at 1:2000 (ThermoFischer Scientific) for additional 1 h at room temperature.