Journal: bioRxiv

Article Title: A stable subgenomic reporter coronavirus enables transcriptional profiling of bystander cells

doi: 10.64898/2026.02.27.708290

Figure Lengend Snippet: Reagents for analysing HCoV-OC43. A. Western blot of Mv.1.Lu cells infected with HCoV-OC43 at an MOI of 10 PFU/cell and harvested at the indicated time points, probed with two anti-Nucleocapsid (N) antibodies, derived from Sheep (Sh) polyclonal antiserum from the MRC PPU & CVR Coronavirus toolkit and a commercial Rabbit (Rb) polyclonal antiserum. Host HSP90 is included as a loading control. B . Immunofluorescence microscopy of HCoV-infected A549 cells, stained with Sheep anti-N and anti double-stranded RNA (dsRNA), a marker of RNA replication. C . Immunoprecipitation (IP) of lysates from HCoV-OC43-infected cells (MOI 3, 24 hpi) using Sheep anti-N and probed with Sheep anti-N and anti-Spike (S). Host HSP90 is included as a loading control. D . Immunofluorescence microscopy of HCoV-OC43-infected A549 cells, stained with mouse monoclonal (Mm) or rabbit polyclonal (Rb) anti-S. E . Silver stain (SS) and western blotting (WB) of virions purified by ultracentrifugation through a 30% sucrose cushion. The supernatant (sup) before and after sucrose cushion, and the pellet, containing purified virions, are shown. Viral structural proteins are indicated by red arrowheads and BSA from cell culture medium by a red asterisk. F . Immunofluorescence microscopy of HCoV-OC43-infected A549 cells stained with anti-SARS-CoV M. Scale bars represent 10 µm.

Article Snippet: Membranes were probed with sheep polyclonal anti-Nucleocapsid anti-serum (MRC PPU & CVR Coronavirus Toolkit, Sheep No. DA116, 1:1000) , rabbit anti-Nucleocapsid (40643-T62, Sino Biological, 1:1000), anti-Spike (PAB21478-100, The Native Antigen Company, 1:1000), anti-SARS-CoV Membrane (ABIN1887462, Antibodies Online, 1:200), anti-MHV nucleocapsid J3.3 ( ) (1:200), anti-GAPDH (AM4300, Invitrogen, 1:4000) and anti-HSP90 (MA5-35624, Invitrogen, 1:2000).

Techniques: Western Blot, Infection, Derivative Assay, Control, Immunofluorescence, Microscopy, Staining, Marker, Immunoprecipitation, Silver Staining, Purification, Cell Culture

Journal: Frontiers in Immunology

Article Title: Vascular Endothelial Growth Factor Receptor, fms-Like Tyrosine Kinase-1 (Flt-1), as a Novel Binding Partner for SARS-CoV-2 Spike Receptor-Binding Domain

doi: 10.3389/fimmu.2022.906063

Figure Lengend Snippet: Demonstrating the binding between rspike RBD and rsFlt-1 using ELISA: (A) Binding of anti-sFlt-1 antibody to spike-RBD immobilized on the ELISA plate, control is without spike RBD immobilization. It confirms normal distribution by both Shapiro-Wilk and Kolmogorov-Smirnov test and significant deviation from zero (P<0.0001) was observed by simple linear regression test. (B) Binding of serial dilutions (0.01,0.1, and 1µg) of rsFlt-1 to increased concentrations of rspike RBD immobilized on the ELISA plate and probed with polyclonal sFlt-1 antibody. Addition of VEGF to the wells immobilized with spike RBD instead of sFlt-1 was used as control. (C) Binding of serial dilutions of rsFlt-1 (0.1, 0.25, 0.5 and 1µg) immobilized on ELISA plate to spike RBD (0.1 µg) which further probed with anti-spike antibody. Control is without sFlt-1 immobilization. One-sample t test (two-tailed) was used to compare between test and control group revealed significant increase in signal upon spike RBD addition (P value =0.0036). (D) Binding of serial dilutions of rsFlt-1 (0.2, 0.5, 1 and 2.5 µg) immobilized on ELISA plate to increased concentrations of spike RBD (0.01, 0.1 and 1µg) which further probed with anti-spike antibody. Wells coated with VEGF were used as control. (E–H) ELISA assays to test the cross-reactivity for rspike RBD, sFlt-1, spike RBD-sFlt-1, or nucleocapsid protein immobilized on the ELISA plates to the human anti-spike polyclonal antibodies (1:10, 1:100, 1:1000 and 1:10000 dilutions) from vaccinated donors (dose 1 and 2) versus non-vaccinated donors as a negative control. Error bars depicted mean SEM ± SD, Statistical test for normal distribution of panels (B, D) using Shapiro-Wilk test confirms normality. Further, two-way ANOVA was used for multiple groups comparison by Tukey and Dunnett’s multiple comparisons tests in panels (B, D) respectively. Panels (E–H) confirms normal distribution by Shapiro-Wilk test followed by two-way ANOVA Dunnett’s multiple comparisons test was applied to compare between control (non-vaccinated) versus1 st and 2 nd dose of vaccinated serum, P-values indicate statistical significance compared to control groups (*P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001).

Article Snippet: Rabbit polyclonal anti-spike RBD HRP tagged secondary antibody, mouse monoclonal anti-spike RBD, Goat anti-human IgG- horseradish peroxidase (HRP) conjugated antibody (Denovo Biolabs Pvt.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Negative Control