Poly prep chromatography column

In vivo psoralen cross-linking, isolation of genomic DNA from mammalian cells, enrichment of replication intermediates, and analysis of data was performed as previously described (Neelsen et al., 2014 (link); Thangavel et al., 2015 (link)). Briefly, 5–10 × 10⁶ Mouse Embryonic Fibroblasts (Rosa^+/ERCRE Atm^+/C) cells were harvested and genomic DNA was cross-linked by three rounds of incubation in 10 µg/ml 4,5′,8-trimethylpsoralen (Sigma-Aldrich) and 3 min of irradiation with 366 nm UV light on a precooled metal block. Cells were lysed and cellular proteins were digested with 2 mg proteinase K (Life technologies) in 5 ml digestion buffer. DNA was purified by isopropanol precipitation, restriction digested with PvuII HF for 4 hr at 37°C, and replication intermediates were enriched using benzolylated naphthoylated DEAE-cellulose (Sigma–Aldrich) in 3 ml BioRad poly-prep chromatography columns. Samples were prepared by spreading DNA on carbon-coated grids in the presence of benzyl-dimethyl-alkylammonium chloride and formamide. Rotary platinum shadowing was performed using the Balzers BAF400 with Quartz crystal thin film monitor. Images were acquired on a JOEL 1200 EX transmission electron microscope with side-mounted camera (AMTXR41 supported by AMT software v601) and analyzed with ImageJ (National Institutes of Health).

The ACE2-Mb TM was purified from cell culture supernatant via its Strep-tag using a Strep-Tactin^®XT buffer set and respective columns (IBA-Lifesciences, Göttingen, Germany) according to the manufacturer´s instructions. For EM purification via its His-tag, cell culture supernatant was added to PBS-equilibrated Ni-NTA agarose (Qiagen, Hilden, Germany) in Poly-Prep^® chromatography columns (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). After two consecutive washing steps with 1 × PBS containing 150 mM NaCl and 10 or 20 mM imidazole, respectively, the EM was eluted using 1 × PBS with 150 mM NaCl and 350 mM imidazole. Isolated Ab constructs were dialyzed over night against 1 × PBS at 4°C and analyzed via SDS-PAGE followed by staining with Quick Coomassie^® Stain (Serva Electrophoresis GmbH, Heidelberg, Germany) or immunoblotting. In the latter case, a Trans-Blot Turbo RTA Mini 0.2 µm Nitrocellulose Transfer Kit (Bio-Rad Laboratories GmbH) and a DIG Wash and Block Buffer Set (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) were used according to the manufacturer’s instructions. For TM detection, the anti-La mAb (5B9) (79 (link)) and an AP-conjugated rabbit-anti-mouse IgG Ab (Dianova GmbH, Hamburg, Germany) were applied.

HDL or LDL/VLDL was removed from HP and pooled normal HS by immunodepletion with anti–apoA-I or anti-apoB antibodies, respectively. For apoA-I depletion, 100 μL HP or HS was incubated with Goat Anti-Human Apolipoprotein AI Sepharose 4B Gel 11A-G1 Resin 1 mL (Academy Bio-Medical, catalog 11A-G1 Resin 1 mL) which was suspended in 400 μL saline and then collected by gravity flow using Poly-Prep Chromatography Columns (Bio-Rad, catalog 7311550) according to the manufacturer’s instructions. ApoB depletion was performed using LipoSep IP (catalog LS-01, Sun Diagnostics) according to the manufacturer’s instructions.

The purified yeast complex I (at a final concentration of 1.78 μM) was mixed with 10-fold excess of GST, GST-Atg38 (1 to 226), GST-Atg38 (1 to 78), or GST-Atg38 (87 to 226). Initially, GST or GST fusion proteins were incubated on ice for 30 min with 20 µL of gluthathione Sepharose 4 Fast Flow resin, which had been equilibrated with reaction buffer (50 mM Tris, pH 8.8, 150 mM NaCl, 1 mM TCEP). The beads were washed once with 1 mL of reaction buffer, then the volume was adjusted to 90 µL. A 10 µL aliquot of 17.8 µM complex I was added to the beads, which were then incubated on ice for 90 min. The reactions were transferred to Poly-Prep Chromatography Columns (Bio-Rad, 7311550), and washed once with 10 mL reaction buffer by gravity flow. The columns were plugged, and 90 µL of reaction buffer was added to the resin to represent the bound fractions. For analysis, samples consisting of either 1 1:10 dilution of the input or the bound fractions were loaded on a SDS-Page gel. After electrophoresis, the gel was stained with InstantBlue (Expedeon, ISB1L).

The production and purification of SARS-CoV-2 NP and SP antigens were performed according to previously described protocols (14 (link), 24 (link), 25 (link)). The RBD of the SP was produced in Expi293F cells as previously described (24 (link), 25 (link)). Rabbit antisera against the RBD and NP were generated at BioGenes GmbH (Berlin, Germany) as follows: day 0 initial dose of 150 μg, day 7 booster of 75 μg, day 14 booster of 75 μg, day 28 booster of 150 μg, and day 42 final bleed. For affinity purification, the RBD and NP were coupled to CNBr-Sepharose 4B (Cytiva) according to the manufacturer’s protocol. The respective antisera were passed through coupled Sepharoses packed into Poly-Prep chromatography columns (Bio-Rad), washed with 20 column volumes of PBS, eluted (0.1 M glycine, 150 mM NaCl [pH 2.5]) with 2 M Tris (pH 9.0), concentrated using an Amicon Ultra 15-ml 100-kDa-NMWL (nominal molecular weight limit) centrifugal filter (Millipore/Merck), and dialyzed against PBS using Slide-A-Lyzer dialysis cassettes (Thermo Scientific).