Anti flag agarose beads

Strains were grown at 25°C overnight to an OD₆₀₀ around 1.0. Seventy OD₆₀₀ units of cells were collected from each strain. Cell lysates were prepared as described previously with modifications (Yue et al., 2017 (link); Liu et al., 2022 (link)). Briefly, cell pellets were washed once in cold water and resuspended in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 5 mM NaF, 1 mM sodium pyrophosphate, and 1 mM DTT) and a protease inhibitor cocktail (Roche). Cells suspensions were transferred to 2 ml screw cap tubes containing prewashed 2 mg zirconia/silica 0.5 mm beads. Cell were lysed using a bead beater; 1% (v/v) Triton X-100 was added to the cell lysates and incubated for 15 min at 4°C. The cell lysates were then spun at 20,000× g for 30 min and supernatants were incubated with 10 µl prewashed anti-Flag agarose beads (Sigma, A2220) at 4°C for 3 hr. Beads were washed five times with lysis buffer containing 0.1% (v/v) Triton X-100. Proteins bound on the beads were eluted with ×1 sample buffer. Proteins were detected with anti-Flag antibody (Sigma, F1804, monoclonal, 1:1000) or anti-Sso antibody (rabbit antiserum, 1:2000).
Coordinates and structure factors for the Sec3-Sso2 complex have been deposited in the Protein Data Bank with accession code 7Q83 (https://www.rcsb.org/structure/7Q83).

All lysates were generated using CHAPS lysis buffer (150 mM KCl, 50 mM Hepes, pH 7.5, 1 mM MgCl₂, 1 mM EDTA, 10% glycerol, 0.5% CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], 5 mM β-mercaptoethanol, and 0.01 μl/ml protease inhibitor cocktail [P8340; Sigma-Aldrich]). For probing, anti-Flag (14793S; Cell Signaling Technology) was used at 1:1,000 dilution. For endogenous controls, anti-GAPH was used at 1:1,000 (5174T; Cell Signaling Technology). Coimmunoprecipitations were performed using anti-Flag agarose beads (M8823; Sigma-Aldrich). In brief, HEK293T lysates containing Flag-POT1 and HA-TPP1 were incubated in BC150 buffer (25 mM Hepes, 2 mM EDTA, 150 mM KCl, and 10% glycerol, pH 7.6) with 20 μl of Flag beads for 6 h at 4°C. Beads were then washed with lysis buffer and BC150 three times. Elution was performed with 0.3 mg/ml Flag peptide. Western blots were run on elutions and probed for Flag and hemagglutinin (HA). Anti-HA was used at 1:1,000 (3724T; Cell Signaling Technology).

2 mg of yeast mitochondria was solubilized with lysis buffer (20 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10% glycerol, 1% digitonin, and complete mini EDTA-free (Roche)). The lysates were incubated with anti-FLAG agarose beads (Sigma-Aldrich) at 4 °C for 2 h. After washing the beads with lysis buffer three times, immunoprecipitates were eluted with 2% SDS. The eluates were then analyzed by Western blotting using antibodies against HA (Santa Cruz), FLAG M2 (Sigma-Aldrich), and Tim23 (A gift from T. Endo, Kyoto Sangyo University, Kyoto, Japan).

For in vivo Co-IP, cells were lysed in lysis buffer (0.5% Triton X-100, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, and 1 mM EDTA) with a fresh protease inhibitor cocktail (Roche). Clarified supernatants were incubated with anti-Flag agarose beads (Sigma) or anti-HA magnetic beads (Pierce) for 4 h at 4°C. Immunoprecipitated complexes were washed three times with lysis buffer that contained 300 mM NaCl and then subjected to immunoblotting with the indicated antibodies.
For in vitro pull-down experiments, recombinant GFP-TRAF6 protein, GFP, and His-KIZ were purified from E. coli. His-KIZ protein was incubated with GFP or GFP-TRAF6 at 4°C for 3 h and then incubated with anti-GFP agarose beads at 4°C for 2 h, washed three times with washing buffer (25 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol) and subjected to immunoblotting.

Human 293T cells were independently transfected with plasmids encoding FLAG-tagged mutant and wild-type hACE2 proteins, FLAG-tagged pACE2 protein, and untagged SARS-CoV/SARS-CoV-2 S protein. For some experiments, cells were treated with tunicamycin (1 μg/ml) for 16 h before harvesting. After 24 h, the cells expressing each ACE2 variant or S protein were lysed in 1 ml of whole-cell extract buffer. Lysates were centrifuged at 14,000 rpm for 30 min at 4°C. Post-spin lysates were then precleared using protein A-agarose (Sigma) for 1 h at 4°C; a small aliquot of each of these lysates was stored as an input sample. Precleared lysates containing the differently tagged or untagged proteins were mixed in a 1:1 ratio and incubated with anti-FLAG-agarose beads (Sigma) overnight at 4°C to precipitate the FLAG-tagged proteins. Beads containing the immunoprecipitate were washed four times in whole-cell extract buffer. Subsequently, immune complexes were eluted using 200 μg of 3× FLAG peptide/ml in whole-cell extract buffer without Triton X-100. The eluted samples were separated by SDS-PAGE and analyzed by Western blotting using anti-FLAG M2 monoclonal antibody or SARS-CoV/SARS-CoV-2 spike protein S2 monoclonal antibody.