rat anti mouse scavenger receptor type i ii cd204  (Bio-Rad)

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: Msr1 contributes to VSV pathogenesis in mice (A) Msr1 mRNA expression in various tissues of wild-type (WT, C57BL/6) and Msr1 −/− mice. LOD, limit of detection; N = 3 mice per genotype. (B and C) The survival curves (B) and disease scores (C) of WT and Msr1 −/− mice challenged with 1×10 7 plaque-forming units (PFUs) per mouse of VSV by retro-orbital injection, N = 12 mice/genotype. p = 0.001 (log rank test), ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, non-parametric Mann-Whitney U test. All the error bars: mean ± standard error of the mean (S.E.M.). (D) The WT mice with paralyzed hindlimbs at day 6 after infection. (E) Immunoblots of Msr1 protein expression in bone-marrow-derived macrophages (BMDMs) of WT, Msr1 −/− , and Msr1 +/− littermates. β-actin is a housekeeping control. (F and G) The survival curves (F) and disease scores (G) of Msr1 −/− and Msr1 +/− littermates challenged with 1 × 10 7 PFU/mouse of VSV by retro-orbital injection, N = 6 mice/genotype. For percent survival, p < 0.01 (log rank test); for disease score, ∗∗p < 0.01, non-parametric Mann-Whitney U test. All the error bars: mean ± S.E.M.

Article Snippet: rabbit anti-human/mouse MSR1 , Origene , Cat# TA336699.

Techniques: Expressing, Injection, MANN-WHITNEY, Infection, Western Blot, Derivative Assay

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: Msr1 is dispensable for systemic VSV dissemination and innate immune responses (A) The RNA viremia in the whole blood of WT and Msr1 −/− mice, Msr1 +/− , and Msr1 −/− littermates infected with VSV, assessed by quantitative RT-PCR, N = 6 mice/genotype in each experiment. The VSV RNA is expressed as a fold change over limit of detection (LOD)s. (B) The serum levels of type I IFN and inflammatory cytokines (IFN-α, IFN-γ, TNF-α, IL-10, CXCL-10, IL-1β, IL-6) in Msr1 +/− and Msr1 −/− littermates after VSV infection, assessed by multiplex ELISA, N = 6 mice/genotype. All the data are presented as mean ± S.E.M., and statistical significance is analyzed by non-parametric Mann-Whitney U tests.

Article Snippet: rabbit anti-human/mouse MSR1 , Origene , Cat# TA336699.

Techniques: Infection, Quantitative RT-PCR, Multiplex Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: Msr1 is critical for VSV infection in the central nervous system (A–C) Quantitative RT-PCR analyses of VSV loads in (A) different tissues of Msr1 +/− and Msr1 −/− littermates on day 6 after infection (N = 3 mice/genotype), (B) the spinal cords, and (C) the brains of Msr1 +/− and Msr1 −/− littermates on day 6 after infection (N = 6 mice/genotype). The viral RNA load is expressed as fold changes over the limit of detection (LOD). (D) The VSV titers in the spinal cord and brain of Msr1 +/− and Msr1 −/− littermates on day 6 after infection, assessed by a plaque-forming assay (N = 6 mice/genotype). (E) Msr1 mRNA expression (expressed as a fold change over the mock spinal cord group) in different tissues of mock and VSV-infected Msr1 +/− mice on day 6 after infection, N = 3 mice/group. (F–H) (F) Correlation between Msr1 expression and VSV load in various tissues after VSV infection, N = 3 mice/group. The mRNA expression of class A and B scavenger receptors in (G) the spinal cord and (H) the brain of mock and VSV-infected Msr1 +/− and Msr1 −/− littermates on day 6 after virus inoculation, assessed by quantitative RT-PCR and expressed as a fold change over the Msr1 +/- -mock group, N = 3 mice/group. (I) Ldlr mRNA expression in the brain and spinal cord of mock and VSV-infected Msr1 +/− and Msr1 −/− littermates, expressed as a fold change over the brain of Msr1 +/- -mock group, N = 3 mice/group, ∗p < 0.05 vs brain, analyzed by non-parametric Mann-Whitney U test. Mock: no virus infection control. (J) Colocalization of MSR1 with microglia, astrocytes, and neurons in the mouse spinal cord by dual immunofluorescence staining. IBA1: a marker of microglia; GFAP: a marker of astrocyte; MAP2: a marker of neuron; DAPI: nuclei. The yellow regions in the overlay indicate colocalizations. Scale bar represents 10 μm. All the data are presented as mean ± S.E.M., and statistical significances are analyzed by non-parametric Mann-Whitney U test, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: rabbit anti-human/mouse MSR1 , Origene , Cat# TA336699.

Techniques: Infection, Quantitative RT-PCR, Expressing, MANN-WHITNEY, Immunofluorescence, Staining, Marker

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: MSR1 mediates cellular entry of VSV in primary mouse cells and human cells (A and B) VSV loads in primary neurons of Msr1 +/− and Msr1 −/− littermates at 36 h after inoculation (MOI = 1, multiplicity of infection = 3), as assessed by (A) quantitative RT-PCR (expressed as a fold change over the lowest viral load) and (B) plaque forming assay. Each dot represents an individual mouse. (C) Immunoblots of MSR1 protein expression in WT and Msr1 −/− bone-marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs). (D–F) (D) VSV-G protein expression in BMDMs, (E) virus titer in culture medium, and (F) VSV-GFP fluorescence under microscopy in BMDMs at 48 h after VSV-GFP infection (MOI = 5), N = 3 biological replicates. Objective: 20×, scale bar: 100 μm. (G) Quantitative RT-PCR analyses of VSV virions attached to BMDMs (4°C for 2 h) and entry into BMDMs (37°C for 30 min). The cells were washed 3 times with cold PBS before switching from 4°C to 37°C and after 37°C as well. MOI = 10, N = 3 biological replicates. (H) GFP expression in WT and MSR1 −/− trophoblasts, 24 h after transduction with GFP-encoding VSV-G-pseudotyped lentiviral vectors (VSV-G-LV-GFP) without polybrene. Scale bar: 50 μm. The statistic VSV-G-LV-GFP was acquired with a fluorescence microscope from 9 random fields of three biological replicates. (I) GFP expression in WT and LDLR −/− + RAP trophoblasts, 24 h after transduction with VSV-G-LV-GFP. The LDLR −/− trophoblasts were pre-treated with RAP (200 nM, 30 min, 37°C) followed by VSV-G-LV-GFP transduction. (J and K) The GFP fluorescence (J) and immunoblots of protein level (K) in VSV-GFP-infected WT and LDLR −/− +RAP trophoblasts with or without MSR1 overexpression; the WT and LDLR −/− trophoblasts were transfected with human MSR1 plasmid for 24 h, followed by VSV-GFP infection for 18 h (MOI = 0.5). The LDLR −/− trophoblasts were pre-treated with RAP (200 nM, 30 min, 37°C) before inoculation of VSV-GFP. Scale bar: 50 μm. (L) Immunofluorescence staining for different Myc-tagged MSR1 fragments in human trophoblasts, 24 h after transfection of plasmids. Myc proteins were stained by a mouse anti-Myc antibody, followed by an Alexa Fluor 488 (green)-conjugated secondary antibody. The cell nuclei were stained by DAPI (blue). Scale bar: 10 μm. β-actin is a housekeeping control. Mock: no virus infection control. All the data are presented as mean ± S.E.M., and statistical significances are analyzed by a standard two-tailed unpaired Student's t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: rabbit anti-human/mouse MSR1 , Origene , Cat# TA336699.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing, Derivative Assay, Fluorescence, Microscopy, Transduction, Over Expression, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Two Tailed Test

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: MSR1 facilitates cellular entry of VSV through its extracellular domains (A–C) WT and MSR1 −/− trophoblasts were examined at 12 h after VSV-GFP inoculation, (A) immunoblots of VSV-G protein level, (B) VSV-GFP fluorescence intensity, and (C) VSV titers in the cell culture medium, MOI = 0.5. N = 3 biological replicates. (D–F) WT and MSR1-overexpressed trophoblasts were examined at 12 h after VSV-GFP inoculation, (D) MSR1 protein expression, VSV-G protein level, (E) VSV-GFP fluorescence intensity, and (F) VSV titers in the culture medium, MOI = 0.5. N = 3 biological replicates. The VSV-GFP fluorescence intensity in (B) and (E) was acquired with a fluorescence microscope from 9 random fields of three biological replicates and quantified by ImageJ. Objective: 4x (top) and 20x, scale bar: 100 μm. (G) Immunoblots of VSV-G protein level in MSR1 −/− trophoblasts with epichromosomal complementation of an empty vector or FLAG-MSR1 expression plasmid at 12 h after VSV infection, MOI=0.5. (H) The protein expression of different FLAG-tagged MSR1 fragments at 24 h after transfection of plasmids in trophoblasts. IB: immunoblotting. Amino residues 1-50 (cytoplasmic N-tail), 1-80 (cytoplasmic N-tail plus transmembrane), 51-end (extracellular domains with transmembrane), and 1-end (full length). (I) VSV-G protein level and (J) VSV-GFP fluorescence intensity in trophoblasts overexpressing FLAG-MSR1 fragments at 12 h after VSV-GFP infection at an MOI = 0.5. The GFP fluorescence intensity was acquired with a fluorescence microscope from random regions of three biological replicates (N = 3). β-actin is a housekeeping control. Mock: no virus infection control. Scale bar: 100 μm. All the data are presented as mean ± S.E.M., and statistical significances are analyzed by a standard two-tailed unpaired Student's t-test, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, compare with WT or vector.

Article Snippet: rabbit anti-human/mouse MSR1 , Origene , Cat# TA336699.

Techniques: Western Blot, Fluorescence, Cell Culture, Expressing, Microscopy, Plasmid Preparation, Infection, Transfection, Two Tailed Test

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: MSR1 interacts with VSV glycoprotein G via its extracellular domains (A–C) Binding of MSR1 to VSV virions. FLAG-MSR1 (human, 1-end) and its fragments (aa1-80, 51-end) were expressed in HEK293T cells and immunoprecipitated (IP) using anti-FLAG antibody-coated magnetic beads, which were then incubated with intact VSV-GFP virions (4°C for 2 h). The bound virions were eluted for (A) immunoblotting (IB) with a mouse monoclonal anti-FLAG and rabbit anti-VSV-G antibody (B and C) Re-infection in Vero cells for 12 h followed by detection of (B) VSV-GFP by fluorescence microscopy and (C) VSV-G protein level by immunoblotting. Mock: no virion control. (D) co-IP of FLAG-MSR1 and its fragments with VSV-G in HEK293T cells transfected with plasmids. WCE: whole-cell extraction. Arrows point to the faint protein bands.

Article Snippet: rabbit anti-human/mouse MSR1 , Origene , Cat# TA336699.

Techniques: Binding Assay, Immunoprecipitation, Magnetic Beads, Incubation, Western Blot, Infection, Fluorescence, Microscopy, Co-Immunoprecipitation Assay, Transfection

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet:

Article Snippet: rabbit anti-human/mouse MSR1 , Origene , Cat# TA336699.

Techniques: Recombinant, Software