rabbit anti vigilin  (Cusabio)

Journal: Nature microbiology

Article Title: An RNA-centric dissection of host complexes controlling flavivirus infection.

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: Fig. 2 | Intersection of ChIRP-MS with genome-wide CRISPR screens nominates functionally relevant pro-viral host proteins. a, Genome-scale CRISPR KO screens of all four DENV serotypes (DENV-1276RKI, DENV-2429557, DENV-3Philippines/H871856 and DENV-4BC287/97) in Huh7.5.1 cells. The genetic screens were independently performed for each serotype, analysed with MAGeCK and combined to obtain the robust rank aggregation (RRA) significance scores (y axis). The 50 most-enriched genes were coloured and grouped by function. b, Scatter plot depicting the enrichment scores of high-confidence ChIRP-MS DENV hits (x axis) and the 200 top-scoring hits from DENV CRISPR genetic screens (y axis). Common hits shared by both the DENV genetic screens and DENV ChIRP-MS are indicated in red (vigilin), blue (RRBP1) and purple (others). c, Western blot analysis of WT and clonal RRBP1-KO (top) or vigilin-KO (bottom) Huh7.5.1 cells. Representative western blot of n = 2 biologically independent replicates showing similar results. d, Analysis by qRT–PCR of WT and RRBP1-KO cells infected with DENV (48 h.p.i.; m.o.i. of 0.1), ZIKVPRVABC59 (48 h.p.i.; m.o.i. of 0.1), POWVLB (48 h.p.i.; m.o.i. of 0.1) or CHIKV181/25 (24 h.p.i.; m.o.i. of 0.01). e, Analysis by qRT–PCR of WT and vigilin-KO cells infected as in d. The WT datasets for POWV in d,e were derived from the same experiments. f, Western blot analysis of the cell lysates of DENV-2429557-infected (72 h.p.i.; m.o.i. of 0.1) WT, RRBP1-KO and vigilin-KO Huh7.5.1 cells probed with antibodies to DENV prM and NS3. Representative western blot of n = 4 biologically independent replicates showing similar results. g, Titres of the infectious particles produced from WT, RRBP1-KO and vigilin-KO Huh7.5.1 cells infected with DENV-2429557 for 72 h at an m.o.i. of 0.1. d,e,g, The data represent the mean ± s.e.m. of n = 3 independent biological replicates, except POWV, where n = 4 independent biological replicates. All P values were determined by two-tailed, unpaired t-tests using GraphPad Prism; n.s., not significant.

Article Snippet: The cells were incubated with antibodies against RRBP1 (Bethyl, A303-996A; 1:100 dilution), vigilin (Bethyl, A303-971A; 1:20 dilution), GFP (Thermo Fisher Scientific, MA5-15256; 1:100 dilution) for 90 min at room temperature.

Techniques: Genome Wide, CRISPR, Western Blot, Quantitative RT-PCR, Infection, Derivative Assay, Produced, Two Tailed Test

Journal: Nature microbiology

Article Title: An RNA-centric dissection of host complexes controlling flavivirus infection.

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: Fig. 3 | RRBP1 and vigilin interact at the ER. a, Single-cell quantification and correlation between RRBP1 or vigilin and the ER–GFP or DAPI immunofluorescence signals. The total numbers of cells that were randomly chosen for each analysis and the mean ± s.e.m. are indicated. b, Western blot analysis of the ER and cytosolic cell fractions probed with GAPDH or tubulin (cytosolic markers), RPN1 (ER marker), RRBP1 and vigilin antibodies. Representative western blot of n = 3 biologically independent replicates showing similar results. c, Western blot analysis of three independent co-IP experiments from uninfected Huh7.5.1 cells with RRBP1 as the bait showing similar results. The samples were treated with or without RNase A. d,e, Representative immunofluorescence of RRBP1 (d) and vigilin (e) co-stained with RNA fluorescent-in-situ-hybridization targeting of DENV or ZIKV positive-stranded RNA genomes. Representative images of n = 2 biologically independent replicates showing similar results. Scale bars, 10 μm. IP, immunoprecipitation.

Article Snippet: The cells were incubated with antibodies against RRBP1 (Bethyl, A303-996A; 1:100 dilution), vigilin (Bethyl, A303-971A; 1:20 dilution), GFP (Thermo Fisher Scientific, MA5-15256; 1:100 dilution) for 90 min at room temperature.

Techniques: Immunofluorescence, Western Blot, Marker, Co-Immunoprecipitation Assay, Staining, In Situ Hybridization, Immunoprecipitation

Journal: Nature microbiology

Article Title: An RNA-centric dissection of host complexes controlling flavivirus infection.

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: Fig. 4 | DENV and ZIKV co-opt the RNA-binding properties of RRBP1 and vigilin in human cells. a, RRBP1 (left) and vigilin (right) irCLIP RT-stop mapping statistics annotated to the human, and DENV and ZIKV genomes (gRNA) and the ribosomal RNAs (rRNA) from Huh7.5.1 cells infected with an m.o.i. of 0.1 for 48 h. b, Histogram of RT stops mapping to the rRNAs from the RRBP1 (top) and vigilin (bottom) irCLIP in uninfected Huh7.5.1 cells. The three cytosolic rRNAs are highlighted. The red dashed line denotes the strongest vigilin binding site, which is adjacent to that of RRBP1. c, Annotation of peaks called from the RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapping to functional elements of human mRNAs including the 5′ UTR, exons, 3′ UTR and introns. The enrichment values were calculated based on the size of each function domain relative to the human genome. d, RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapped at base resolution to the DENV genome. The RT stop intensity was normalized to the total number of unique reads mapping to the viral genome. The 5′ and 3′ UTR regions are highlighted in red and blue, respectively.

Article Snippet: The cells were incubated with antibodies against RRBP1 (Bethyl, A303-996A; 1:100 dilution), vigilin (Bethyl, A303-971A; 1:20 dilution), GFP (Thermo Fisher Scientific, MA5-15256; 1:100 dilution) for 90 min at room temperature.

Techniques: RNA Binding Assay, Infection, Binding Assay, Functional Assay