Ab9108

His Tag antibody plates were coated with a 0.5 µg/mL solution of spike protein antigens diluted in PBS at 4 °C for 12 h then washed with 1X TBST. To test blocking of ACE2 binding, 100 µL of precipitated phage (10¹³ titer) or 10 µg/mL of converted antibody was applied to each well and incubated for 1 h at 4 °C. After vigorous washing, the wells were then incubated with 100 µL of recombinant ACE2-His protein (RayBiotech Life, Inc, Peachtree Corners, GA) for 1 h at 4 °C. After 6X washing, 100 µL of rabbit anti-6X His tag polyclonal antibody (Abcam, ab9108) diluted 1:1,000 in PBS was added and incubated for 1 h at 4 °C. Following 6 washes with 1X TBST, wells were then incubated with 100 µL of goat anti-rabbit IgG (H + L) antibody HRP diluted 1:10,000 in 1X TBST for 1 h at 4 °C. Competitive ELISAs were developed and measured identical to binding ELISAs described above. A negative control antibody, SARS-CoV-2 spike mouse mAb (40591-MM42) (Sino Biological, Wayne, PA) that binds to the SARS-CoV-2 spike protein but does not neutralize, and a positive control antibody, SARS-CoV-2 spike mouse mAb (40592-MM57) (Sino Biological) that blocks interaction with ACE2 were used at 10 µg/mL. An ACE2-His alone condition without phage or antibody served as the baseline signal for spike-ACE2 binding. Blocking potential was measured as a decrease in signal from ACE2-His alone.

The coding region of Ghd8 was cloned into the pET-32a vector (Novagen) and pGEX-6P-1 vector (GE Healthcare) with EcoRI and XhoI sites, respectively. To obtain the OsHAP5b protein, we used the same method for GHD8. The recombinant expression vector was expressed in Escherichia coli Transetta (DE3) cells (Transgen). The pull-down experiment was performed as described previously [50 (link)]. In brief, supernatants with equal amounts of Glutathione S-transferase (GST) or OsHAP5b-GST with GHD8-His recombinant proteins were incubated for 6 h at 4 °C in a total volume of 2 ml of pull-down buffer (20 mmol Tris-HCl, pH 8.0, 200 mmol NaCl, 1 mmol EDTA, 0.5% Lgepal CA-630 and protease inhibitor), after which 200 μl of GST resin was added (GE Healthcare; 17–5132-01) and the mixture incubated for 2 h at 4 °C. The binding reaction was then washed 5 times (10 min each time at 4 °C) using the pull-down buffer. After extensive washing, the pulled-down proteins were eluted by boiling at 95 °C for 10 min, separated on 12% SDS-PAGE and detected by immunoblots using an anti-GST antibody (Abcam; ab19256) and anti-His antibody (Abcam; ab9108), respectively.

HUVECs at a cell density of 1 × 10⁴ cells/well in a 96-well plate were fixed with 4% PFA at room temperature for 10 min. Non-permeabilized cells were blocked with 3% BSA/PBS at room temperature for 2 h before they were treated with various concentrations of TS5-p45 at room temperature for 2 h. Following the treatment, cells were washed with PBST thrice at room temperature for 5 min each and incubated with anti-His (Abcam, ab9108) and isotype control anti-IgG antibody (Santa Cruz, sc2027) incubations at 4 °C overnight. The next day, cells were washed as described and incubated with goat anti-rabbit IgG, Alexa Fluor 488 (Invitrogen, A-11034) at room temperature for 2 h, before fluorescence intensity was measured with Hidex Sense Microplate Reader (Hidex, Finland). Next, cells were counter-stained with 1 µg/ml DAPI (Calbiochem, Sigma-Aldrich, 268298) in PBS at room temperature for 10 min, followed by washing as described. Fluorescence intensity measurements for DAPI were used to normalize TS5-p45 fluorescence intensity across all conditions.

Anti-FLAG antibody-coated plate (GenScript, L00455C), recombinant His-tagged TS5-p45, and FLAG-tagged NCL-FL and Ctrl overexpression WCL were used in this experiment. Specific FLAG-tagged NCL-FL capture was first verified by titrating the Anti-FLAG antibody-coated plate with increasing doses of NCL-FL lysate. Anti-NCL primary antibody (Abcam, ab22758) and Alexa Fluor 488 conjugated anti-rabbit IgG (Invitrogen, A-11034) were used for detection. The same protocol was applied to Ctrl lysates as a negative control. Saturation binding experiment was then performed by titrating TS5-p45 over FLAG-tagged NCL-FL- and Ctrl-captured wells at 4 °C for overnight incubation. The following day, treated wells were incubated with anti-His (Abcam, ab9108) at room temperature for 2 h and Alexa Fluor 488 conjugated anti-rabbit IgG (Invitrogen, A-11034) at room temperature for 1 h. Finally, fluorescence intensity was measured with Hidex Sense Microplate Reader (Hidex, Finland). Binding affinity was calculated with GraphPad Prism 8.

Proteins were transferred to a nitrocellulose membrane using iBLOT system (Life Technologies). A rabbit anti-Histag (ab9108, Abcam,UK) or anti-acfC antibody were used as primary antibodies and a fluorescently labelled goat anti-rabbit IR Dye 680RD secondary antibody (Li-cor) was used as secondary antibody. The membrane was visualized using an Odyssey fluorescent imager (LI-COR). The un-cropped image of Fig. 1b is in Supplementary Figure 1.