polyclonal rabbit anti phospo syk ab  (Santa Cruz Biotechnology)

Journal: Mucosal Immunology

Article Title: Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model

doi: 10.1038/s41385-019-0167-z

Figure Lengend Snippet: Human CD89 is functional in Tg mice. a Flow-cytometric analysis of CD89 expression and mIgA binding on CD89 tg/wt /LyzM cre/wt (black) and littermate (filled grey) bone marrow-derived macrophages (BMDM). Fluorescence-minus-one (FMO) control is shown in open grey line. Right: Binding of fluorescently labelled polyclonal mIgA is restricted to the mCherry low population. b Flow-cytometry binding studies of various human IgA species. BMDM from CD89 tg/wt /LyzM cre/wt (black) and littermates (grey) were incubated with polyclonal human mIgA and IgAM preparations and monoclonal human IgA1 and IgA2. Bound IgA was detected with αIgA-PE. Geometric MFI ± SD from mCherry low-expressing cells from duplicates are shown. c CD89 is associated constitutively with dimeric FcRγ-chain. CD89 was immunoprecipitated (IP) with an anti-CD89 antibody (clone A59) from whole-cell extracts (right panel) prepared from unstimulated BMDM from a CD89 tg/wt /LyzM cre/wt mouse. Protein was eluted and run on SDS-PAGE under non-reducing conditions. The membrane was probed with an anti-FcRγ chain antibody. d CD89 mediates induction of Syk and p42/44 phosphorylation. CD89 tg/wt /LyzM cre/wt and littermate (LM) BMDM cells were incubated with IgA-coated beads for the indicated times. Samples were run on SDS-PAGE and Syk and p42/44 phosphorylation were determined using phospho-specific antibodies. The same membrane was reprobed with anti-Syk and anti-p42/44 antibodies. e CD89 facilitates uptake of IgA-coated beads. CD89 tg/wt /LyzM cre/wt (black) and littermate (grey) BMDMs cultured in eight-well slides were incubated at 37 °C with IgA- or IgG-coated beads at a cell-to-bead ratio of 25:1 for 30 min. Slides were stained with HOECHST and anti-kappa-PE to visualise nuclei and external beads and imaged. The number of internalised beads per cell were counted manually. Over 100 BMDM cells per stimulus were counted. The percentage of cells with internalised numbers of beads are shown

Article Snippet: Phosphorylated Syk was detected with polyclonal rabbit anti-phospo-Syk Ab (sc-293118, Santa Cruz) and with HRP-conjugated goat anti-rabbit Ab (P0448, DAKO).

Techniques: Functional Assay, Expressing, Binding Assay, Derivative Assay, Fluorescence, Control, Flow Cytometry, Incubation, Immunoprecipitation, SDS Page, Membrane, Phospho-proteomics, Cell Culture, Staining

Journal: Mucosal Immunology

Article Title: Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model

doi: 10.1038/s41385-019-0167-z

Figure Lengend Snippet: Human CD89 mediates cytokine production by BMDM. a BMDM from CD89 tg/wt /LyzM cre/wt (black) and littermates (grey) were cultured for 24 h on surface-bound polyclonal mIgA, IgAM or IgG. Cytokine concentrations in pg/ml (mean ± SEM, n = 3 individual mice) are shown. The effect of the CD89 protein was tested using a two-way ANOVA with Sidak’s multiple comparison test. b CD89 tg/wt /LyzM cre/wt BMDM were incubated overnight with mIgA-coated (●) or BSA-coated beads (▲) at varying ratios. Cytokine concentrations in pg/ml (means ± SD) from duplicates are shown

Article Snippet: Phosphorylated Syk was detected with polyclonal rabbit anti-phospo-Syk Ab (sc-293118, Santa Cruz) and with HRP-conjugated goat anti-rabbit Ab (P0448, DAKO).

Techniques: Cell Culture, Comparison, Incubation

Journal: Mucosal Immunology

Article Title: Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model

doi: 10.1038/s41385-019-0167-z

Figure Lengend Snippet: Reactivity of human polyclonal Igs to Cal7 and PR8 H1N1 strain

Article Snippet: Phosphorylated Syk was detected with polyclonal rabbit anti-phospo-Syk Ab (sc-293118, Santa Cruz) and with HRP-conjugated goat anti-rabbit Ab (P0448, DAKO).

Techniques:

Journal: Mucosal Immunology

Article Title: Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model

doi: 10.1038/s41385-019-0167-z

Figure Lengend Snippet: Polyclonal human Ig preparations inhibit Cal7 ( a , c ) and PR8 ( b , d ) H1N1 influenza neuraminidase activity and virus plaque formation in vitro. a , b Neuraminidase activity assay. IC 50 concentrations are shown in brackets. c , d MDCK virus plaque neutralisation assay. Error bars indicate SD

Article Snippet: Phosphorylated Syk was detected with polyclonal rabbit anti-phospo-Syk Ab (sc-293118, Santa Cruz) and with HRP-conjugated goat anti-rabbit Ab (P0448, DAKO).

Techniques: Activity Assay, Virus, In Vitro

Journal: Mucosal Immunology

Article Title: Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model

doi: 10.1038/s41385-019-0167-z

Figure Lengend Snippet: Human polyclonal mIgA protects mice from PR8 H1N1-induced death in a CD89-dependent manner. In total, 2.5 mg of Ig preparation was administered i.n. as indicated in ( a ) and mice were infected i.n. with 500 PFU of PR8 H1N1 on day 0. b Percentage of body weight of CD89 tg/wt /LyzM cre/wt (black) and littermates (grey) treated with IgG (square), mIgA (circle) or PBS (diamond). c Minimum weight as a percentage of day 0. Mean ± SEM from two pooled experiments ( n = 5–15 mice). The shaded area indicates mice euthanised at the humane endpoint. Unpaired t test was performed to compare CD89 tg/wt /LyzM cre/wt and littermates

Article Snippet: Phosphorylated Syk was detected with polyclonal rabbit anti-phospo-Syk Ab (sc-293118, Santa Cruz) and with HRP-conjugated goat anti-rabbit Ab (P0448, DAKO).

Techniques: Infection

Journal: Mucosal Immunology

Article Title: Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model

doi: 10.1038/s41385-019-0167-z

Figure Lengend Snippet: Human polyclonal Igs protect from weight loss and IgG reduces Cal7 and PR8 H1N1 viral titres in vivo. In total, 2.5 mg of Ig preparation was administered i.n. to CD89 tg/wt /LyzM cre/wt mice and mice infected i.n. with H1N1 influenza on day 0 as indicated in Fig. 7a. Lung viral titres at day 5 ( a , b ) and weight ( c , d ) after i.n. inoculation of 10 4 PFU Cal7 ( a , c ) and 500 PFU PR8 H1N1 ( b , d ) on day 0 are shown. Mean ± SEM values from one experiment are shown ( n = 5) for each virus. One-way ANOVA with Dunn’s multiple comparison to the PBS group is shown. ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Phosphorylated Syk was detected with polyclonal rabbit anti-phospo-Syk Ab (sc-293118, Santa Cruz) and with HRP-conjugated goat anti-rabbit Ab (P0448, DAKO).

Techniques: In Vivo, Infection, Virus, Comparison

Journal: Mucosal Immunology

Article Title: Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model

doi: 10.1038/s41385-019-0167-z

Figure Lengend Snippet: Human polyclonal Igs reduce influenza-induced transcriptional profile and protein levels in the lung. mRNA transcriptional levels ( a , b ) and protein cytokine levels ( c , d ) of homogenised lungs at day 5 after i.n. inoculation of 10 4 PFU Cal7 ( a , c ) and 500 PFU PR8 ( b , d ) H1N1 and Ig treatment as indicated in Fig. are shown. CNRQ, calibrated normalised relative quantities from n = 3–5 animals are shown for each experiment. Mean protein concentrations from one experiment are shown ( n = 4–5) for each virus. One-way ANOVA with Dunnet’s multiple comparison to the PBS group is shown. ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.d. not done

Article Snippet: Phosphorylated Syk was detected with polyclonal rabbit anti-phospo-Syk Ab (sc-293118, Santa Cruz) and with HRP-conjugated goat anti-rabbit Ab (P0448, DAKO).

Techniques: Virus, Comparison

Journal:

Article Title: Differential Role of the Fas/Fas Ligand Apoptotic Pathway in Inflammation and Lung Fibrosis Associated with Reovirus 1/L-Induced Bronchiolitis Obliterans Organizing Pneumonia and Acute Respiratory Distress Syndrome 1

doi: 10.4049/jimmunol.0901958

Figure Lengend Snippet: The presence of both caspase-8 and caspase-3 are diminished in zIETD-fmk reovirus 1/L-induced ARDS or BOOP. CBA/J mice were i.n. inoculated with either 1 × 106 PFU (BOOP) or 1 × 107 PFU (ARDS) reovirus 1/L and were left untreated or treated with 5 mg/kg of either zVAD-fmk or zIETD-fmk daily beginning on day 3 postinoculation until sacrifice. A, Lungs of reovirus 1/L-induced BOOP and ARDS on day 14 postinoculation were paraffin embedded and stained with a rabbit polyclonal Ab for active/cleaved caspase-8 in either untreated (top) or zIETD-fmk-treated (bottom) reovirus 1/L-iduced BOOP or ARDS. IHC for active/cleaved caspase-8 is representative of two experiments with two mice per time point. B, Western analysis from whole lung lysates for protein expression of cleaved caspase-3 was determined in reovirus 1/L-induced ARDS on day 9 postinoculation and in reovirus 1/L-induced BOOP on day 14 postinoculation. Relative expression of cleaved caspase-3 was determined by comparing the ratio of the cleaved caspase-3 band to background. Histograms represent densitometric data from the mean ± SD autoradiogram signals from three mice for the saline, ARDS-induced, and BOOP-induced mice and from at least four mice in the zVAD-fmk- and zIETD-fmk-treated groups. *, p < 0.05 compared with saline-inoculated, control mice; **, p < 0.05 compared with reovirus 1/L-inoculated mice.

Article Snippet: Western blotting was then accomplished using either the rabbit anti-mouse Fas polyclonal Ab (A20; Santa Cruz Biotechnology), the mouse antimouse FasL mAb (Kay10; BD Pharmingen), or the rabbit anti-caspase-3 antiserum (H-277, 1/500 dilution; Santa Cruz Biotechnology) with the appropriate secondary Abs.

Techniques: Staining, Western Blot, Expressing, Saline, Control

Journal:

Article Title: Differential Role of the Fas/Fas Ligand Apoptotic Pathway in Inflammation and Lung Fibrosis Associated with Reovirus 1/L-Induced Bronchiolitis Obliterans Organizing Pneumonia and Acute Respiratory Distress Syndrome 1

doi: 10.4049/jimmunol.0901958

Figure Lengend Snippet: The presence of both caspase-8 and caspase-3 are diminished in zIETD-fmk reovirus 1/L-induced ARDS or BOOP. CBA/J mice were i.n. inoculated with either 1 × 106 PFU (BOOP) or 1 × 107 PFU (ARDS) reovirus 1/L and were left untreated or treated with 5 mg/kg of either zVAD-fmk or zIETD-fmk daily beginning on day 3 postinoculation until sacrifice. A, Lungs of reovirus 1/L-induced BOOP and ARDS on day 14 postinoculation were paraffin embedded and stained with a rabbit polyclonal Ab for active/cleaved caspase-8 in either untreated (top) or zIETD-fmk-treated (bottom) reovirus 1/L-iduced BOOP or ARDS. IHC for active/cleaved caspase-8 is representative of two experiments with two mice per time point. B, Western analysis from whole lung lysates for protein expression of cleaved caspase-3 was determined in reovirus 1/L-induced ARDS on day 9 postinoculation and in reovirus 1/L-induced BOOP on day 14 postinoculation. Relative expression of cleaved caspase-3 was determined by comparing the ratio of the cleaved caspase-3 band to background. Histograms represent densitometric data from the mean ± SD autoradiogram signals from three mice for the saline, ARDS-induced, and BOOP-induced mice and from at least four mice in the zVAD-fmk- and zIETD-fmk-treated groups. *, p < 0.05 compared with saline-inoculated, control mice; **, p < 0.05 compared with reovirus 1/L-inoculated mice.

Article Snippet: The slides were then immersed in 2% hydrogen peroxide to quench endogenous peroxidase for 10 min and incubated with 5% normal goat serum (Vector Laboratories) for 1 h. For IHC for Fas analysis, incubation with a rabbit anti-mouse Fas polyclonal Ab (A20, 1/100 dilution; Santa Cruz Biotechnology) for 30 min was performed followed by incubation with biotinylated anti-rabbit IgG (1/200 dilution; Santa Cruz Biotechnology) for 30 min. IHC for FasL analysis was performed using the M.O.M.

Techniques: Staining, Western Blot, Expressing