Figure S5.

Expression, localization, and interaction analyses for virus and host proteins, related to Figure 5

(A) Scatter plot displaying average log₂ fold changes in SND1 coIP experiments (x axis) and average log₂ fold changes in total proteome measurements of input lysates used for coIP experiments (y axis). SARS-CoV-2 infected cells are compared relative to uninfected cells in coIPs and total proteome measurements (n = 2 independent infections). Proteins that display a significant interaction and expression change (absolute log₂ fold change > 1, FDR < 0.05) upon SARS-CoV-2 infection cells are highlighted.

(B) IF staining of SND1 and NSP3 proteins in SARS-CoV-2 infected A549^ACE2 cells at 8 hpi (MOI = 10 PFU/cell). Mock-infected cells were stained as controls. Representative images are shown. Overlap between NSP3 and SND1 is quantified by Manders’ co-localization coefficient (right, n = 12 images). Scale bars, 30 μm.

(C) Log₂ fold changes for viral proteins observed in total proteome measurements of SARS-CoV-2 infected A549^ACE2 cells at different infection time points, as previously reported.¹⁸

(D) As in (B), but for SND1 and NSP9. Overlap between NSP9 and SND1 is quantified by Manders’ co-localization coefficient (right, n = 10 images).

(E) Microscale thermophoresis (MST) assay to monitor binding of recombinant NSP9 protein to recombinant SND1 protein in vitro. Unlabeled NSP9 protein (254 pM to 8.33 μM) was titrated against site-specific cysteine-labeled SND1 (10 nM) and thermophoresis was recorded. Change in fluorescence (ΔFnorm) was measured at MST on-time of 10 s. Values are mean ± SD (n = 3 independent measurements).

(F) CoIP western blot analysis for epitope-tagged SND1 and NSP9 proteins exogenously expressed in uninfected HEK293T cells. SND1-FLAG constructs are deleted for indicated protein domains. NSP9-V5 served as bait. Input lysates are shown on the left. Anti-FLAG and anti-V5 antibodies are used to detect tagged SND1 and NSP9 proteins, respectively. SN, SNase.