## 3 Results  
Murine neuroblastoma Neuro2a (N2a) cells are known to show high protein expression owing to their high plasmid DNA transfection efficiency. Since N2a cells have high protein expression efficiency, N2a cells were selected as a cell line for SARS-CoV-associated protein expression. To establish a strategy for protein purification from N2a cells, subcellular localization of transmembrane-lacking hACE2, S1, and S1-2 subunits tagged with fluorescent proteins in N2a cells was confirmed using confocal fluorescence microscopy. The hACE2-eGFP (ACE2), ER-mCherry-S1 (S1), and ER-mCherry-S1-2 (S1-2) were observed inside the subcellular structures, including the ER (Figure 1A). Some ER-mCherry foci were colocalized with lysotracker green (LysoG Figure 1B). Since monomeric red fluorescent protein (mRFP) and its variants (e.g., mCherry) are often observed in lysosomes--mainly because of their low p\(K_{\rm a}\)[27; 28]--these results suggest that ACE2, S1, and S1-2 are efficiently expressed and localized in various secretory pathways, including the ER and lysosome.  
The concentrations of ACE2, S1, and S2 in cell lysates determined using FCS were \(<\)10 nM; thus, ACE2, S1, and S1-2 were purified and concentrated from the lysates. Western blotting using tag-specific antibodies revealed the successful recovery of intact proteins (Figure 2A). Contaminants of purified samples were confirmed by SDS-PAGE followed by silver staining (Figure 2B). However, these purified samples were used for the measurements because FCCS can specifically analyze target molecular interactions, even in the presence of non-fluorescent contaminants.  
Figure 1: Confocal fluorescent images of murine neuroblastoma Neuro2a cells expressing eGFP-tagged hACE2 and mCherry-tagged S1 proteins. (**A**) Fluorescence images of Neuro2a cells expressing hACE2-eGFP, ER-mCherry-S1, or ER-mCherry-S1-2. Cells co-expressing hACE2-eGFP and ER-mCherry as an ER localization marker, \(a\); ssYFP-BiP and ER-mCherry-S1, \(b\); ssYFP-BiP and ER-mCherry-S1-2, \(c\). Bar = 5 \(\upmu\)m. (**B**) Lysotracker Green-stained Neuro2a cells expressing ER-mCherry as control, ER-mCherry-S1, and ER-mCherry-S1-2. Bar = 10 \(\upmu\)m.  
The positive amplitude of normalized cross-correlation functions (nCCFs) indicates the interaction between two-colored fluorophores [25; 29]; thus, typical nCCFs were compared when ACE2 was mixed with S1, S1-2, or mCherry monomers. The amplitude of nCCF of the mixture between ACE2 and mCherry monomers as a control was not observed (Ctrl in Figure 3A). In contrast, the amplitudes of nCCFs of the mixture between ACE2 and S1 or S1-2 were positive (green or magenta lines in Figure 3A). Furthermore, the amplitude between ACE2 and S1-2 was higher than that between ACE2 and S1 (Figure 3A). To quantitatively compare the interaction strengths among them, the relative cross-correlation amplitude (RCA), which is the ratio of the number of interacting molecules to the total number of green-fluorescent molecules, was calculated. RCAs of the mixture between eGFP or mCherry monomers and ACE2, S1, or S1-2 as controls were almost zero; however, the RCAs of the mixture between ACE2 and S1 or S1-2 were significantly positive (Figure 3B). RCA in the case of S1-2 was higher than that in S1 (Figure 3B). These results suggest that both S1 and S1-2 interact with ACE2, and that S1-2 interacts more strongly than S1. Moreover, counts per molecule (CPM) values--the mean brightness of a single molecule--of eGFP- or mCherry-tagged proteins were not different from those of eGFP or mCherry monomers, suggesting that ACE2, S1, and S1-2 existed as monomers in the mixture (Figure 3C,D).  
Figure 2: Confirmation of purified recombinant hACE2-eGFP, ER-mCherry-S1, and ER-mCherry-S1-2. (**A**) Western blot of purified recombinant eGFP, hACE2-eGFP, ER-mCherry, ER-mCherry-S1, and ER-mCherry-S1-2 using anti-GFP and anti-mCherry antibodies (left and right, respectively). (**B**) SDS-PAGE gel followed by silver staining of purified hACE2-eGFP, ER-mCherry-S1, and ER-mCherry-S1-2.