Assays

Cells (4x 15 cm dishes) were harvested and lysed with 3 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P40 (NP40) and EDTA-free protease inhibitor cocktail tablets). Centrifugation-cleared lysates (13,000 rpm) were filtered through 0.45 µm spin filters (Millipore Ultrafree-CL) and immunoprecipitated with 60 µl anti-HA resin. Resin containing immune complexes were washed five times with lysis buffer followed by five PBS washes, and elution with 150 µl of 250 mg/ml HA peptide in ...

HeLa cells stably expressing UBE2QL1-APEX2 were grown in lysine- and arginine-free DMEM supplemented with FBS, L-Glutamine, Sodium pyruvate, heavy arginine (R10) (38 μg/ml) and lysine (K8) (66 μg/ml) or light arginine (R0) (38 μg/ml) and lysine (K0) (66 μg/ml), respectively. Further experiments were conducted as soon as the cells reached a protein labelling with heavy amino acids of at least 95%. Heavy-labeled cells were treated with 250 μM Leu-Leu methyl ester hydrobromide (LLOMe, Sigma) for 3 ...

Frozen cell pellets from 4x15 cm cell culture plates were lysed in Glycerol buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.5 % Triton-X-100, 10 % Glycerol, 1x protease inhibitor, 1x phosphatase inhibitor) for 30 min at 4° C with end-over-end rotation. Lysates were cleared from cell debris by centrifugation prior to adjustment of protein concentrations between the samples and overnight immunoprecipitation at 4° C with pre-equilibrated anti-HA-agarose (Sigma). Agarose beads were washed five ...

Cells were incubated with 500 µM Biotin-Phenol during the last 30 min of LLOMe or GPN treatment and subsequently pulsed by addition of H2O2 for 1 min at room temperature. To stop the biotinylation reaction, they were washed 3x with quencher solution (10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox in DPBS) and 3x with PBS. All further steps were performed at 4°C unless indicated otherwise. After cell harvest with 0.25% Trypsin/EDTA (ThermoFisher Scientific), cells were counted and heavy- ...

APEX2-mediated biotinylation of cells was carried out as described before (Hung et al., 2016). In brief, cells were supplemented with 500 µM biotin-phenol (IrisBiotech) for 30 min at 37°C before addition of 1 mM H2O2 at room temperature. Cells were then first washed with quencher solution (1 mM sodium azide, 10 mM sodium ascorbate and 5 mM Trolox in DPBS), then with DPBS, scraped and harvested. All steps were carried out at 4°C unless stated otherwise. Cells were washed and suspended in homogenization ...

Samples were essentially prepared as described in Zellner et al. Mol Cell 2021 (DOI: 10.1016/j.molcel.2021.01.009)

Sample preparation for LC-MS/MS Eight million of Cy3/5-positive autophagosomes (roughly 10ug of proteins) were denatured with 2% sodium deoxycholate, 50 mM Tris-HCl pH 8.5, 2.5 mM TCEP, 10 mM chloroacetamide at 95°C for 10 min. Lysates were prepared with in-StageTip (iST) processing method for LC-MS/MS as previously described by Kulak et al. 2014. Briefly, Proteins were digested overnight at 37°C with 1 volume of 50mM Tris-HCl pH 8.5 containing LysC (Wako Chemicals) at 1:100 (w/w) ratio and Trypsin ...