cleaved sars cov 2 spike ser686  (Cell Signaling Technology Inc)

Journal: International journal of molecular sciences

Article Title: SARS-CoV-2 Spike Protein Mutation at Cysteine-488 Impairs Its Golgi Localization and Intracellular S1/S2 Processing.

doi: 10.3390/ijms232415834

Figure Lengend Snippet: Figure 3. Defect of S1/S2 processing in C488 mutant spike proteins. (A) Spike-expressing plasmid was transfected into VeroE6/TMPRSS2 cells, and the cell lysate was prepared 20 h post-transfection. Full-length and Ser-686 cleaved spike proteins were probed with anti-spike (1A9) and cleaved SARS- CoV-2 spike (Ser686) antibodies, respectively. GAPDH is shown as a loading control. (B) Wild-type spike-expressing plasmid was transfected in HEK293 cells, which were treated with CCF642 for 13 h at 5 h post-transfection, followed by cell lysate preparation. Full-length and Ser-686 cleaved spike proteins were detected with anti-spike (1A9) and cleaved SARS-CoV-2 spike (Ser686) antibodies, respectively. (C) Each mutant spike-expressing plasmid was transfected in NCI-H522/hACE2 (left panels) and HEK293T (right panels) cells. After 20 h, cell lysates were prepared, and full-length and Ser-686 cleaved spike proteins were detected with anti-spike (1A9) and cleaved SARS-CoV-2 spike (Ser686) antibodies, respectively.

Article Snippet: Cleaved SARS-CoV-2 spike protein (Ser686) antibodies (Cat#84534) and anti-mouse IgG–HRP (Cat#7076) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Transfection, Control

Journal: International journal of molecular sciences

Article Title: SARS-CoV-2 Spike Protein Mutation at Cysteine-488 Impairs Its Golgi Localization and Intracellular S1/S2 Processing.

doi: 10.3390/ijms232415834

Figure Lengend Snippet: Figure 4. M protein did not affect C488A-mutant spike. (A) VeroE6/TMPRSS2 cells were co- transfected with spike-, M-, N-expressing plasmids with pEGFP-C1. The cells were fixed at 20 h post-transfection, and the green fluorescence protein (GFP) signals were photographed with conven- tional fluorescence microscopy. Areas of GFP-positive green cells were measured in each picture by ImageJ software, and average area size of GFP-positive cells in a picture was plotted in the right graph. One-way ANOVA with Tukey-HSD was performed to assess statistical significance. n.s. means “not significant”, and * indicates p < 0.01. (B) Spike-expressing plasmids were co-transfected with SARS-CoV-2 M or N protein-expressing plasmid in Vero cells, which were fixed and permeabilized. Co-localization of spikes (green) with GM130 (red) were analyzed by confocal microscopy. White arrows indicate complete co-localization (yellow) of spikes with GM130. (C) Co-localization of spikes and GM130 was examined in the transfected cells (n ≥156), and the ratio of co-localization is shown. One-way ANOVA with Tukey-HSD was performed to assess statistical significance. n.s. means “not significant”, and * indicates p < 0.01. (D,E) SARS-CoV-2 spike-expressing plasmid was co-transfected with M- or N-expressing plasmid in HEK293T cells. The cell lysate was prepared after 20 h, and full-length and Ser-686 cleaved spike proteins were detected by Western blotting with anti-spike (1A9) and cleaved SARS-CoV-2 spike (Ser686) antibodies, respectively. Triplicated transfected samples were analyzed (Exp. 1–3), and signal intensity was quantified by ImageJ (right graph in E).

Article Snippet: Cleaved SARS-CoV-2 spike protein (Ser686) antibodies (Cat#84534) and anti-mouse IgG–HRP (Cat#7076) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Mutagenesis, Transfection, Expressing, Microscopy, Software, Plasmid Preparation, Confocal Microscopy, Western Blot

Journal: International journal of molecular sciences

Article Title: SARS-CoV-2 Spike Protein Mutation at Cysteine-488 Impairs Its Golgi Localization and Intracellular S1/S2 Processing.

doi: 10.3390/ijms232415834

Figure Lengend Snippet: Figure 5. Brefeldin A (BFA) interferes with SARS-CoV-2 spike protein function. (A) Vero cells were transfected with spike-expressing plasmid. The cells were treated with or without 1 µM BFA for an additional 15 h at 9 h post-transfection. The spike protein localization in permeabilized cells was examined using confocal microscopy. The numbers #1 and #2 indicate two independent images. White arrowheads indicate plasma membrane localization of spike protein. (B) HEK293T cells were transfected with spike-expressing plasmid. The cells were treated with 1 µM BFA and 1 µM CID- 1067700 (CID) for additional 19 h at 9 h post-transfection. Full-length and Ser-686 cleaved spike proteins were detected by Western blotting with anti-spike (1A9) and cleaved SARS-CoV-2 spike (Ser686) antibodies, respectively. (C) VeroE6/TMPRSS2 cells were transfected with spike-expressing plasmid with pEGFP-C1. The cells were treated with BFA or CID-1067700 for an additional 15 h at 5 h post-transfection. The green fluorescence protein (GFP) signals and phase contrast cell morphologies were photographed with conventional fluorescence microscopy.

Article Snippet: Cleaved SARS-CoV-2 spike protein (Ser686) antibodies (Cat#84534) and anti-mouse IgG–HRP (Cat#7076) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Clinical Proteomics, Membrane, Western Blot, Microscopy