Journal: Cell Reports

Article Title: SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization

doi: 10.1016/j.celrep.2021.109017

Figure Lengend Snippet:

Article Snippet: The upper portion of the membrane was probed with anti-HA tag antibody (mouse, Sigma-Aldrich, H3663) diluted 1:1,000 in 5% skim milk solution, while the lower portion of the membrane was probed with anti-VSV matrix protein antibody (Kerafast, EB0011; loading control) diluted 1:2,500 in 5% skim milk solution.

Techniques: Produced, Recombinant, Plasmid Preparation, Software, Imaging

Journal: Molecular Cell

Article Title: A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

doi: 10.1016/j.molcel.2020.04.022

Figure Lengend Snippet: The Multibasic S1/S2 Site of SARS-2-S Is Cleaved by Furin, and Cleavage Is Required for Syncytium Formation and Entry into Human Lung Cells (A) Overview of the SARS-S and SARS-2-S S1/S2 mutants analyzed. (B) Analysis of furin-mediated S protein priming. Rhabdoviral particles harboring the indicated S proteins containing a C-terminal V5 tag for detection were lysed and subjected to western blot analysis. Detection of vesicular stomatitis virus matrix protein (VSV-M) served as control. (C) Rhabdoviral particles bearing MERS-S, SARS-S, or SARS-2-S equipped with a V5 or HA epitope tag at their C terminus (or no glycoprotein at all, control) were produced in the absence or presence of furin inhibitor (FI, decanoyl-RVKR-CMK; 1 μM or 10 μM) and analyzed for S protein processing by western blot analysis. Detection of VSV-M served as control. (D) Syncytium formation assay: Vero or Vero-TMPRSS2 cells were transfected to express the indicated S proteins (or no S protein, empty vector, control). At 24 h post transfection, cells were incubated in the presence or absence of trypsin (1 μg/mL) for an additional 24 h before they were fixed, stained with May-Gruenwald and Giemsa solution, and analyzed by bright field microscopy (scale bars, 200 μm). White arrowheads indicate syncytia. For (B)–(D), representative data from three (B and C) or four (D) independent experiments are shown. (E) Transduction of Vero (TMPRSS2 − ) and Calu-3 (TMPRSS2 + ) cells with rhabdoviral particles bearing the indicated S proteins or vesicular stomatitis virus glycoprotein (VSV-G). At 16 h post transduction, virus-encoded firefly luciferase was quantified in cell lysates. Presented are the mean data from three independent experiments. Transduction efficiency is shown relative to that measured for particles not bearing a viral glycoprotein. Error bars indicate the standard error of the mean. Statistical significance was tested by one-way analysis of variance with Dunnett’s post test (p > 0.05, ns; ∗∗∗ p ≤ 0.001).

Article Snippet: The blots were then incubated over night at 4°C with primary antibody solution (all antibodies were diluted in PBS-T containing 5% skim milk; mouse anti-HA tag [Sigma-Aldrich, H3663, 1:2,500], mouse anti-V5 tag [Thermo Fisher Scientific, R960-25, 1:2,500] or VSV matrix protein [Kerafast, EB0011, 1:2,500]).

Techniques: Western Blot, Produced, Tube Formation Assay, Transfection, Plasmid Preparation, Incubation, Staining, Microscopy, Transduction, Luciferase

Journal: Molecular Cell

Article Title: A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

doi: 10.1016/j.molcel.2020.04.022

Figure Lengend Snippet:

Article Snippet: The blots were then incubated over night at 4°C with primary antibody solution (all antibodies were diluted in PBS-T containing 5% skim milk; mouse anti-HA tag [Sigma-Aldrich, H3663, 1:2,500], mouse anti-V5 tag [Thermo Fisher Scientific, R960-25, 1:2,500] or VSV matrix protein [Kerafast, EB0011, 1:2,500]).

Techniques: Produced, Recombinant, Plasmid Preparation, Software, Imaging