Assays

Parental and ITCH KO HeLa cells were cultured in lysine- and arginine-free DMEM supplemented with dialyzed FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin and light (K0) lysine (38 μg/mL) and arginine (66 μg/ml). Heavy medium was the same except the light lysine was replaced with K8-lysine (L-Lysine, 2HCl U-13C U-15N, Cambridge Isotope Laboratories Inc). All cells were treated for 1 h with 1mM LLOMe (Sigma). Subsequently, cells were processed as described before (Fiskin et ...

Blood samples (20 ml) from clinically affected homozygous Npc1 mutation carriers and healthy donors were collected. Negative selection of peripheral blood monocyte-derived macrophages was performed by incubating full blood for 20 min at RT with RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies). An equal volume of washing buffer (D-PBS/2% FBS/1 mM EDTA) was added to each sample and layer of macrophages was separated from red blood cells and plasma by centrifugation on a Ficoll ...

Samples were processed and analyszed as described in Behrends et al., Nature 2010.

Anti-HA-immunoprecipitation was performed as previously described (Behrends et al, 2010; Jung et al, 2015; Jung et al, 2017; Sowa et al, 2009). Summarily, expression of TAPL-HA and coreTAPL-HA was induced by addition of 4 µg/ml doxycycline for 24 h in HeLa Flp-In T-REx cells. Parental non-transfected HeLa Flp-In T-REx cells were used as negative control. For each sample, 6.4 x 107 cells were harvested, frozen in liquid nitrogen and stored at -80 °C. Cells were lysed in 3 ml MCLB buffer (50 mM ...

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 ...