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: Nature Communications

Article Title: RNA replicon vaccination confers long-lasting protection against H5N1 avian influenza in 23 zoo bird species

doi: 10.1038/s41467-025-64301-5

Figure Lengend Snippet: a Genome maps of VSV and VSVΔG(H5) vector. VSV contains five genes (N, P, M, G, and L). In VSVΔG(H5), the G gene is replaced by the HA gene of A/Dalmatian Pelican/Bern/1/2022 (H5N1) encoding either a polybasic (pb) or monobasic (mb) cleavage site. b Infectious virus yield on BHK-G43 helper cells infected with VSVΔG(H5 mb ) at m.o.i. 0.05 f.f.u./cell. At 20 h p.i., virus particles were concentrated from 200 mL of cell culture supernatant by ultracentrifugation (UC) and resuspended in 20 mL PBS. Infectious titers before and after concentration were determined on BHK-21 cells. The infectious titers of n = 12 vaccine batches are shown (blue triangles). The height of the bars indicates the geometric mean value. The error bars show the 95% confidence interval. Statistical significance was tested by the unpaired, two-sided Student’s t test (df = 22; t = 4.732). c Western blot of VSVΔG(H5 pb ) and VSVΔG(H5 mb ) particles after ultracentrifugation. Viral proteins were separated by SDS-PAGE under reducing conditions, stained with colloidal Coomassie (left panel), or immunostained with chicken polyclonal anti-H5 serum. The relative molecular mass (in kDa) according to the migration of molecular weight markers is indicated on left hand side. A representative experiment out of two performed is shown. d Surface H5 antigen was detected with bovine α-H5 serum. VSV matrix (M) protein was stained in permeabilized cells using mAb 23H12 (α-VSV M). VSV*ΔG-infected cells visualized by GFP. Nuclei stained with DAPI. Scale bar = 20 μm. A representative experiment out of two performed is shown. e Multicycle virus replication on MDCK cells. Cells were infected with the indicated viruses (m.o.i. = 0.0001) and cell culture supernatant sampled at 1, 24, and 48 h p.i. Infectious titers were determined on BHK-21 cells (mean ± SD of 3 infection experiments). The detection limit (25 f.f.u. mL -1 ) is indicated (dashed line). The two-way ANOVA with Tukey’s multiple comparison test compared VSVΔG(H5 P :N1 P :GFP) to VSVΔG(H5 pb ) and VSVΔG(H5 mb ). P values are indicated in the graph. f Immunofluorescence analysis of MDCK cells at 20 h p.i. with the indicated viruses (m.o.i. = 0.02). Cells infected with VSVΔG(H5 P :N1 P :GFP) were detected by GFP fluorescence. Cells infected with either VSVΔG(H5 mb ) or VSVΔG(H5 pb ) were detected by indirect immunofluorescence using a monoclonal antibody directed to the VSV M protein. Cell nuclei were stained with DAPI. The bar is equivalent to 100 μm. A representative experiment out of three performed is shown. Source data are provided as a Source Data file.

Article Snippet: The cells were washed with PBS, permeabilized for 5 min with 0.25% (vol/vol) of Triton X-100 in PBS and then incubated for 1 h with a monoclonal antibody directed to VSV matrix (M) protein (mAb 23H12, KeraFast, Boston, MA, cat. no. EB0011), diluted 1:25 in PBS.

Techniques: Plasmid Preparation, Virus, Infection, Cell Culture, Concentration Assay, Western Blot, SDS Page, Staining, Migration, Molecular Weight, Comparison, Immunofluorescence, Fluorescence

Journal: Nature Communications

Article Title: Wipi3 is essential for alternative autophagy and its loss causes neurodegeneration

doi: 10.1038/s41467-020-18892-w

Figure Lengend Snippet: a Schematic diagram of the experiment. VSVG–GFP-expressing Atg5 KO MEFs and Atg5 KO /Wipi3 Cr MEFs were treated with or without etoposide (10 µM) at the restrictive temperature (40 °C) for 12 h. Cells were then shifted to the permissive temperature (32 °C), and fixed at the indicate times. b Representative images at 0, 15, and 60 min after the temperature shift are shown. Golgi were stained with an anti-GS28 antibody, and the location of VSVG-GFP and Golgi were analyzed. Scale bars = 10 µm. c Reduction of PM-localized VSVG–GFP upon etoposide treatment in Atg5 KO MEFs. The Atg5 KO MEFs containing VSVG-GFP were or were not treated with etoposide, and at 60 min after the temperature shift, PM-localized VSVG-GFP was stained with an anti-VSVG antibody, and its amount was measured using flow cytometry. d The indicated MEFs containing VSVG-GFP were treated with or without etoposide (10 µM) in the presence of E64d and pepstatin. At 60 min after the temperature shift, cells were immunostained with an anti-Lamp2 antibody. Bars = 10 µm. Magnified images of the dashed squares are shown in the insets. Bars = 1 µm.

Article Snippet: The following antibodies were used for the immunoblot and immunofluorescence assays: anti-GFP (Santa Cruz Biotechnology, #sc-9996), anti-Wipi3 (Thermo Fisher Scientific, #PA5-50864), anti-Atg5 (Sigma, #A0731), anti-Lamp2 (Abcam, #ab13524), anti-RFP (MBL, #M204-3), anti-Flag (MBL, #PM020), anti-HA (Santa Cruz Biotechnology, #sc-7392), anti-Lamp1 (abcam, #ab24170), anti-VSVG (KeraFAST, #EB0010), anti-GS28 (BD, #611184), anti-GM130 (BD, #610823), anti-Tom20 (Santa Cruz Biotechnology, #sc-11415), anti-Myc (Santa Cruz Biotechnology, #sc-40), anti-Rab9 (Sigma, #R5404), anti-His (Nacalai Tesque, #04428-26), anti-LC3 (nanoTools, #0231-100), anti-p62 (MBL, #PM045), anti-Wipi2 (abcam, #ab105459), anti-actin (Millipore, #MAB1501), anti-GST (Santa Cruz Biotechnology, #sc-138), anti-Atg7 (CST, #2631), anti-GAPDH (EMD Millipore, #MAB374), anti-calbindin D-28k (Swant, #300), anti-calbindin D-28k (Sigma, #C2724), anti-GFAP (Cell Signaling Technology #12389), anti-Iba1 (Wako, #019-19741), anti-ubiquitin (CST, #3933), anti-ceruloplasmin (BD, #611488), anti-ferritin (abcam, #ab69090), anti-ferroportin (Novus, #NBP1-21502) and anti-DMT1 (Proteintech, #20507-1-AP).

Techniques: Expressing, Staining, Flow Cytometry