Vero E6 cells were seeded in 24-well plates and incubated at 37 °C overnight. HTNV, rVSV, or rVSV-HTNV-GP was used to infect cells for 2 h, followed by medium change to DMEM containing 2% FBS and cultured for an additional 48 h. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min, permeabilized with 0.5% Triton X-100 for 10 min, and stained with 1A8 (HTNV NP) (1:1000), anti-Gn-1 (1:50), anti-Gc-10 (1:100), or anti-VSV-G (1:1000, abcam, 309106) antibodies at 4 °C overnight. After washing, Cy3-conjugated goat anti-mouse IgG (1:400, Sangon Biotech, D111024) or Cy3-conjugated goat anti-rabbit IgG (1:400, Sangon Biotech, D111018) were added to each well and incubated at room temperature for 1 h. The nuclei were visualized using Hoechst 33258 (1:1000, YEASEN, 40729ES10). The images were captured using a BX60 fluorescent cell imager (Olympus, Tokyo, Japan).
Anti vsv g
Manufactured by Abcam
Anti-VSV-G is a primary antibody that specifically binds to the Vesicular Stomatitis Virus Glycoprotein (VSV-G) epitope. It is commonly used as a tag for protein expression and detection in various cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 conjugated goat anti mouse igg, by Sangon (1 mentions)
Cy3 conjugated goat anti rabbit igg, by Sangon (1 mentions)
Hoechst 33258, by Yeasen (1 mentions)
Bca protein assay, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse irdye 800, by LI COR (1 mentions)
Goat anti rabbit irdye 680, by LI COR (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Anti gapdh, by Beyotime (1 mentions)
Anti calnexin, by Cell Signaling Technology (1 mentions)
Anti calreticulin, by Cell Signaling Technology (1 mentions)
Anti pdi, by Cell Signaling Technology (1 mentions)
Anti cd19, by Cell Signaling Technology (1 mentions)
Anti cd19 pe, by Beckman Coulter (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Anti actin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Abcam (1 mentions)
Anti flag, by Merck Group (1 mentions)
Goat anti rabbit igg, by Abcam (1 mentions)
4 protocols using anti vsv g
1
Immunofluorescent Staining of Hantavirus Infection
2
Western Blot Protein Detection Protocol
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Cells were lysed with RIPA lysis buffer (Cat. No. sc-24948A; Santa Cruz, USA) and total protein concentrations were determined by the BCA protein assay (Cat. No. 23225; Pierce Rockford, IL, USA). Equal concentrations of harvested protein were electrophoresed on 10% (w/v) sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto a 0.45 μm PVDF membrane (Cat. No. 03010040001; Millipore, Billerica MA, USA) which was activated in methanol. The primary antibodies used to detect the target proteins were as following: anti-VSV-G was purchased from Abcam (Cat. No. ab183497; Cambridge, UK). Anti-RSV-G and anti-GFP were both purchased from Sino Biological (Cat. No. 13029-T52; Cat. No. 13105-R208; Beijing, China). Anti-FOLR1 and anti-APOBEC3B were purchased from ABclonal (Cat. No. A15672; Cat. No. A9010; Wuhan, China). Anti-GAPDH was purchased from Beyotime (Cat. No. AF0006; Shanghai, China). IRDye 680 goat-anti-rabbit and IRDye 800 goat-anti-mouse were purchased from Li-COR (Cat. No. 926-68071; Cat. No. 926-32216; Lincoln, NE, USA), and co-incubated with the membrane. Then they were visualized by a Li-COR Odyssey Infrared Imager. The protein bands were quantified using ImageJ (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, university of Wisconsin, America).
3
Comprehensive Antibody Detection Protocol
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The following antibodies were used for protein detection: anti-CD19–PE (Beckman Coulter; IM1285U), anti-CD19 (Cell Signaling; 3574), anti-CD19 3G7 (Origen; TA506234), anti-CD19 HD37 (Thermo Fisher; MA1-34005), anti-VSVg–fluorescein isothiocyanate (FITC) (Abcam; ab3863), anti-VSVg (Abcam; ab50549), anti-CD81 (LifeSpan; LS-C359239), anti-CD81–FITC (Molecular Probes; A15753), anticalnexin (Cell Signaling; 2679), anticalreticulin (Cell Signaling; 12238), anti-UGCGL1 (NovoPro Laboratories; 116554), anti-PDI (Cell Signaling; 3501), anti-mouse antibody–Alexa Fluor 488 (Thermo Fisher; R37114), and anti-rabbit antibody–Alexa Fluor 594 (Thermo Fisher; A-21207).
4
Protein Immunoblotting Procedure
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The protein samples were prepared as previously described [19 (link)]. In brief, the membranes transferred with the protein samples were blocked with 5% skim milk for 1 h at room temperature. The membrane was incubated overnight at 4 °C with the individual primary antibodies (anti-Flag (1:1000, M2, Sigma), anti-porTrim26 (1:1000, generated in this study), anti-VSV G (1:1000, Abcam), anti-PRRSV N (1:1000, generated by a synthetic peptide of N), and anti-actin (1:10,000, Sigma)). The membrane was then incubated for 1 h at room temperature with the secondary antibodies: horseradish-peroxidase-conjugated goat anti-mouse IgG (1:5000, Abcam) or goat anti-rabbit IgG (1:10,000, Abcam).
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