goat serum amyloid a1 a2 saa1 saa2  (R&D Systems)

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Model of allergen-induced airway hyperresponsiveness (AHR) for wild-type (WT) and Saa–/– mice (both C57BL/6 background). Mice were sensitized i.t. on day 0 (1 μg) and i.n. on days 7–11 with 10 μg of HDM extract. Airway measurements were performed 72 h after the last allergen challenge (used in Fig. 1a-​-jj and Extended Data Fig. 2 and ​and3).3). (b) For SAA1 antibody blockade, we used an established mouse model of allergen-induced AHR sensitizing WT BALB/cJ mice i.t. on day 0 and 14 with 100 µg of HDM extract + isotype control, or HDM + αSAAab. Airway measurements and tissue harvests were performed 72 h after the last allergen challenge (used in Extended Data Fig. 4). (c) In short-term exposure protocols WT and Saa–/– mice (both C57BL/6 background) received a single HDM challenge (100 μg) were sacrificed 16 h later (used in Fig. 2a-​-d).d). (d) In short-term exposure protocols BALB/cJ mice received a single HDM challenge (100 μg)+ isotype control, HDM challenge + HDL (200μg) and isotype control, or HDM + αSAAab and were sacrificed 24 h later (used in Fig. 2e). Contol mice received either PBS + isotype or PBS + HDL and isotype control. (e) For overexpression of SAA1 in vivo, mice were injected 20 µg of DNA complexed to polyethylenimine at day 0, exposed to PBS or HDM 48 h later and ILC2s as well as BAL cytokines measurements were performed on day 3 (used Fig. 2f). (f) WT and Saa–/– mice were sensitized i.t. on day 0 (1 μg) and i.n. on days 7–11 with 10 μg with extracts from the parasitic worm Schistosoma mansoni (a Puerto Rican isolate). Tissues were harvested 72 h after the last allergen challenge (used in Fig. 4). (g) For FPR2 blockade (WRW4, 2 mg/kg), WT BALB/cJ mice were sensitized and challenged i.t. on day 0 and 14 with 100 μg of HDM extract. Airway measurements were performed 72 h after the last allergen challenge (used in Fig. 7a-​-f).f). (h) In short term exposure experiments, BALB/cJ mice received a single HDM challenge (100 μg) or HDM + WRW4 and were sacrificed 24 h later (used in Fig. 7g and ​andi).i). (i) Model of Alternaria alternata (Alt a )-induced airway inflammation for WT and Saa–/– mice. Mice were sensitized i.t. on day 0 (1 μg) and i.n. on days 7–11 with 10 µg of Alt a extract. Tissues were harvested 72 h after the last allergen challenge (used in Extended Data Fig. 9f-​-jj).

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Over Expression, In Vivo, Injection

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: Basal (a) SAA1 and (b) FPR2 mRNA expression in nasal epithelial cells of CRS patients and matched control donors. (c) HDM (100 μg/ml)-triggered IL-33 concentration in primary nasal epithelial cells of CRS patients as compared to controls. (d) Dissociation of hexameric SAA was analyzed separating lipid-free and lipid-bound SAA by HDL pull down using a polyclonal goat antibody specific for human ApoA1 and immunoblotting with a monoclonal mouse antibody specific for human SAA1 (Acris). (e) Bar graph represents quantitative analysis of SAA monomer band intensities (LI-COR Image Studio Software). (f) SAA1 protein amounts in sera of control donors and HDM allergic individuals. Data represents means ± SEM of (a and b) n=16 control and n=27 CRS, (c) n=6 control and n=12 CRS patients per group, (e) n=5 control and n=8 CRS patients per group and (f) n=18 control and n=27 HDM allergic patients per group. Representative immunoblot of one control and one CRS patient (d). Cropped images are shown. P values were calculated with a two-tailed test using Student’s t-test with Welch’s correction. ****P ≤ 0.0001.

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Expressing, Concentration Assay, Western Blot, Software, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) AHR (*P < 0.0105), (b) total serum IgE concentrations (*P = 0.0265), (c) eosinophil infiltration into the lungs, and (d) PAS stained lung sections of isotype (iso), HDM+isotype (HDM), or HDM+αSAAab-treated (αSAA) WT BALB/c mice. Antibodies were administered at 5 μg/i.t. Frequency of (e) Lin-CD45+ST2+IL-13+ ILC2s (**P = 0.0027, ***P = 0.0002), (f) TH2 and (*P = 0.0260, ***P = 0.0001) (g) TH17 cells (*P = 0.0149, ***P = 0.0005) in the lungs of these mice. Data represents means ± SEM of pooled data from 2 independent experiments containing (a) n=6 PBS+iso, n=7 HDM+iso, n=9 HDM+αSAA animals per group; (b, c, f, g) n=9 PBS+iso, n=11 HDM+iso, n=13 HDM+αSAA animals per group or are representative of 2 independent experiments with (e) n=4 PBS+iso, n=5 HDM+iso, n=7 HDM+αSAA animals per group. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s post hoc analysis that compares HDM to iso and αSAA counterparts. ****P ≤ 0.0001

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Neutralization, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: Increase of the type 2 cytokines (a) IL-33 (*P = 0.0138), (b) IL-25, and (c) TSLP in the BAL of WT and Saa–/– mice 24h after a single dose of PBS or HDM. (d) Numbers of IL-13+ ILC2 cells in the lungs of these mice. (e) Effects of local SAA1 neutralization in WT BALB/c mice through HDL (*P = 0.0117) or local SAA antibody blockade (αSAA, *P = 0.0392) of WT BALB/c receiving isotype (iso), HDM+isotype (HDM+iso), or HDM+αSAAab-treated (HDM+αSAA). (f) Effects of SAA1 overexpression on numbers of Lin-CD45+CD25+ST2+IL-13+ ILC2s in the lungs. For overexpression, WT mice were injected retro-orbitally with a SAA1 overexpression plasmid (SAA1, grey bars) or a non-coding control vector (pcDNA, open bars). Data represent means ± SEM of pooled data from 2 independent experiments containing (a-c) n= 6 WT PBS, n=8 WT HDM, n=7 Saa–/– PBS and n=10 Saa–/– HDM animals per group; (d) n= 5 WT PBS, n=9 WT HDM, n=6 Saa–/– PBS and n=9 Saa–/– HDM animals per group; (e) n=7 PBS+iso, n=6 PBS+iso/HDL, n= 11 HDM+iso, n=13 HDM+iso/HDL, n=12 HDM+αSAA animals per group; (f) n=3 pcDNA PBS, n=9 pcDNA HDM, n=3 SAA1 PBS and n=10 SAA1 HDM animals per group. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s post hoc analysis (a-e) or two-sided Student’s t-test (f). ***P ≤ 0.001

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Neutralization, Over Expression, Injection, Plasmid Preparation, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) HDM, rBlo t 13, rDer p2 or rDer p 23 were separated by SDS-PAGE followed by detection with rSAA1 and a mouse monoclonal antibody specific for human SAA1. (b) Migration of SAA1 (1 mg/ml) in PBS was analyzed in the presence of increasing amounts of the mite FABP Blo t 13 (4:1 (0.25 mg/ml), 2:1 (0.5 mg/ml), 1:1 (1 mg/ml) and 1:2 (2 mg/ml) of Blo t 13) by native PAGE followed by immunoblot analysis using mouse monoclonal antibody specific for human SAA1 (R&D systems; MAB30196). (c) SAA1 (1 mg/ml) was chemically cross-linked in the presence of Blo t 13 added at a ratio of 1:2 or 1:4 (as indicated) using 0.0025%, 0.005%, and 0.01% glutaraldehyde and analysed by immunoblot using sequence-specific (amino acid 14–30) rabbit antiserum for human SAA1 (densitometric analysis shown in supplementary table 1). (d) HDM-induced IL-33 concentrations in BEAS-2B cells after siRNA-mediated silencing of SAA1 (siSAA1) or non-targeting scrambled siRNA (siNT) (**P = 0.0002, ns = 0.1911). (e) IL-33 amounts induced by individual mite or unrelated major cat (Fel d 1) and birch pollen (Bet v 1) allergens (10 μg/ml) (. (f) Blo t 13-induced IL-33 in BEAS-2B cells with siRNA-mediated silencing of SAA1 (siSAA1) or transfected with non-targeting scrambled siRNA (siNT). (g) IL-33 amounts induced by Der p 13-depleted HDM extract and HDM extract where Der p 13 was neutralized (100 μg/ml). (h) BAL IL-33 levels (*P = 0.0209, ***P = 0.006) as well as (i) Lin-CD45+ST2+IL-13+ ILC2s in the lungs of WT BALB/c mice receiving a single i.t. challenge with 100 μg Der p 13-depleted HDM extract as compared to isotype-treated control extract (*P = 0.0423). IL-33 amounts induced in the BEAS-2B cell line and SAA1 binding by the human fatty acid binding proteins (j) FABP5 (**P = 0.0207) and (k) FABP7. Cropped images are shown. Data are shown as means ± SEM and are pooled data from 2 (d, e, g, j) or 3 (h, i) independent experiments or representative of 2 (k) or 3 independent experiments (f) each containing at least n=4 biologically independent samples or n=8 PBS, n=12 HDM-isotype and n= 14 HDM-α-group 13 (h) and or n=10 PBS, n=13 HDM-isotype and n= 14 HDM-α-group 13 (i) animals per group. Immunoblots are representative of an experimental n=2 (a-c, j, k). P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (d, e, f), Dunett’s (h) or Holm-Sidaks (i) post hoc analysis, and two-tailed Student’s t-test with Welch’s correction (g, j, k) ****P ≤ 0.0001.

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: SDS Page, Migration, Clear Native PAGE, Western Blot, Sequencing, Transfection, Binding Assay, Two Tailed Test, Comparison

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Migration of SAA1 (1 mg/ml) in IMDM media was analyzed in the presence of the mite FABP rBlo t 13 (1 mg/ml) by native PAGE followed by immunoblot analysis using a sequence-specific antiserum (amino acid 89–104) raised against human SAA1. (b) Effects of the protein synthesis inhibitor cycloheximide on IL-33 concentrations in BEAS-2B cells treated for 30 min with HDM (100 μg/ml). (c) Cell viability of BEAS-2B cells after HDM exposure over time as measured by continuous reduction of a cell viability substrate by viable cells (**P = 0.0041). SAA1 concentrations in cells after (d) siRNA-mediated silencing of SAA1 (siSAA1) or non-targeting scrambled siRNA (siNT). Effects of SAA1 on HDM-induced IL-6 (**P = 0.0086, ***P = 0.0007) and IL-8 release in cells with siRNA mediated knockdown of SAA1 (siSAA1) (e and f). (g) HDM-induced IL-6 amounts after Der p 13-depletion and/or neutralization. Data are shown as means ± SEM and are representative of 2 (b, e) or 3 (c) independent experiments or pooled data from 2 independent experiments (d, f) each containing at least n=4 biologically independent samples. Representative analysis of SAA1 migration patterns in the presence of Blo t 13 using sequence-specific rabbit antiserum raised against human SAA1 (aa 89–104) (a). IL-6 amounts were analysed for one representative experiments with n=5 biologically independent samples (g). Cropped images are shown. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) followed by Dunett’s post (b) or Tukey’s hoc analysis (e,f), two-way ANOVA followed by Dunnet correction (c) or Student’s t-test (d, g). ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Derivative Assay, Migration, Clear Native PAGE, Western Blot, Sequencing, Neutralization, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Sequence of SAA1. Indicated are: secondary structure α-helices (α 1–4) and loops (connecting lines between α-helices), C-terminal tail (CTL). Amino acids located within the hydrophobic core of the SAA1 hexamer are shaded in green. The epitopes recognized by affinity-purified IgGs raised against residues 27–44, 40–63, 68–84, and 89–104 are indicated by yellow lines (used in panel e). The mutation site at the hydrophobic core is indicated by an orange dot (used in panel f). C-terminal deletion of amino acids (Δ1–11) is shaded in blue. (b) BEAS-2B supernatants were cleared of lipid-bound SAA1 pulling down HDL-bound SAA1 using a polyclonal goat anti-ApoA1 antibody. Cleared supernatants were immunoblotted with a monoclonal mouse anti-human SAA1 antibody (Acris). (c) IL-33 secretion induced by rSAA1 alone (open bars) or in complex with a mouse monoclonal antibody specific for human SAA1 (αSAA ab, closed bars) in the BEAS-2B cell line. (d) Supernatants of cells left either untreated (media) or stimulated with recombinant SAA1 (rSAA) and immunoblotted with a monoclonal antibody specific for human SAA (R&D Systems; MAB30196). (e) IL-33 release from BEAS-2B cells after incubation with sequence-specific rabbit antisera specific for human SAA1 (**P = 0.0029). (f) HDM-triggered IL-33 release in BEAS-2B cells transfected with empty plasmid control (EV), wildtype SAA1 (WT) overexpression plasmid or a SAA1 plasmid with a Trp to Ala mutation at position 53 of the amino acid sequence (W53A) to mutate the hydrophobic core of SAA1 (WT to EV **P = 0.0032; WT to W53A **P = 0.0085). (g) HDM-induced IL-33 amounts in epithelial cells grown in media supplemented with charcoal-stripped FBS (**P = 0.0027). Cropped images are shown. Data shown as means ± SEM represent are representative of 2 independent experiments (c, d, f, g) or 3 (e) each containing at least n=4 biologically independent samples. Blots are representative of 2 (d) and 4 (b) independent experiments. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (e, g) or Dunett’s post hoc analysis (f). ****P ≤ 0.0001

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Sequencing, Affinity Purification, Mutagenesis, Recombinant, Incubation, Transfection, Plasmid Preparation, Over Expression, Two Tailed Test, Comparison

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) mRNA and (b) protein amounts of SAA in response to HDM (100 μg/ml). (c) SAA1 hexamer after rBlo t 13 stimulation was analyzed as described in Fig. 3b. (d) Bar graph represents quantitative analysis of SAA hexamer using LI-COR Image Studio Software. (e) Concentration-dependent IL-33 release from BEAS-2B cells induced by rBlo t 13. Data are shown as means ± SEM and are pooled data from 2 independent experiments (b, e) each containing n=4–5 replicate wells or representative of 2–3 independent experiments (d). SAA1 mRNA expression, normalized to the average of housekeeping genes, is presented as mean value ± SEM (n = 5). Immunoblot is representative of an experimental n=2. Cropped images are shown. P values were calculated with a two-tailed test using Student’s t-test (a), two-way analysis of variance (ANOVA) followed by Dunnet’s correction (b) or one-way ANOVA with Dunett’s post hoc analysis (d, e). ****P ≤ 0.0001.

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Software, Concentration Assay, Expressing, Western Blot, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) mRNA expression of the FPR family members FPR1, FPR2 and FPR3 at baseline (open bars) or after 2 h of HDM stimulation (filled bars). (b) HDM-triggered IL-33 amounts in BEAS-2B cells overexpressing human FPR1 (**P = 0.0068, ***P = 0.0003). (c) IL-33 secretion in BEAS-2B cells overexpressing human FPR2 or cells transfected with an empty vector (EV; pcDNA3.1) (**P = 0.0068, ***P = 0.0003). HDM-induced IL-6 and IL-8 amounts in BEAS-2B cells overexpressing FPR2 (d and e) or blocking the FPR2 receptor (f (**P = 0.0043) and g (**P = 0.0021)) using WRW4. Data presented as means ± SEM and is representative of 2 independent experiments each containing at least n= 4 biologically independent samples (b, d, f) or pooled data from 2 independent experiments (c, e, g). mRNA expression, normalized to the average of housekeeping genes, is presented as mean values ± SEM (n = 5 biologically independent samples) performed in duplicates (a). P values were calculated with a two-tailed test using Student’s t-test (a) or one-way analysis of variance (ANOVA) with Tukeys multiple comparison test (b-e) or Dunett’s post hoc analysis (f, g). ****P ≤ 0.0001

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Expressing, Transfection, Plasmid Preparation, Blocking Assay, Two Tailed Test, Comparison

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: IL-33 concentrations in BEAS-2B cells blocking the SAA-binding receptors (a) FPR2 (WRW4, 12 μM; ***P = 0.0006) and (b) TLR4 (LPS from Rhodobacter sphaeroides; LPS-RS, 10 μg/ml; ). (c) Effects of FPR2 or TLR4 blockade in cells transfected with empty plasmid control (EV) or SAA1 overexpression plasmid. **P = 0.0047 § indicates p = 0.056; # indicates p = 0.22 between EV and SAA plasmid. Blockade of (d) CD36 (anti-human CD36 blocking antibody, aCD36; 10 μg/ml; ***P = 0.0005), (e) the P2 receptor antagonist suramin (100 μM; *P = 0.028), and (f) TLR2 (anti-human TLR2 IgA, aTLR2, 10 μg/ml; **P = 0.0055). (g) HDM-triggered IL-33 release in BEAS-2B cells transfected with EV, WT SAA1 overexpression plasmid or a SAA1 plasmid with a deletion of the C-terminal amino acids 1–11 (Δ1–11; *P = 0.01, **P = 0.0078 ). Data are representative of 2–3 independent experiments (b, d-f) or pooled data from 2 independent experiments (a, c, d, g) each containing at least n=4 biologically independent samples and depicted as means ± SEM. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s (a, b, d-g) or Tukey’s (c) post hoc analysis.****P ≤ 0.0001.

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Blocking Assay, Binding Assay, Transfection, Plasmid Preparation, Over Expression, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Immunoblot of SAA1 after Alternaria alternata (Alt a) stimulation of BEAS-2B cells performed as described in Fig. 3b. Alt a-induced IL-33 and IL-8 (**P = 0.0044) secretion in BEAS-2B cells after siRNA-mediated silencing of SAA1 (siSAA1) (b and c) or WRW4-mediated FPR2 blockade (d and e). (f) Total serum IgE concentrations, (g) eosinophil counts and frequency of (h) CD3+CD4+, (i) TH2 and (j) TH17 cells in the lungs of PBS or Alt a-treated WT and Saa–/– mice. Immunoblots are representative of an experimental n=2 (a). Data are presented as means ± SEM and represent pooled data from 2 independent experiments (b, d, e, f, g, i, j) or show one representative experiment (c) each containing at least n= 4 biologically independent samples or n=8 WT PBS, n=13 WT Alt a, n=8 Saa–/– PBS and n=11 Saa–/– Alt a animals per group (f, g, i, j) or n=5 WT PBS, n=9 WT Alt a, n=5 Saa–/– PBS and n=7 Saa–/– Alt a animals per group (h). Cropped images are shown. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Tukeys multiple comparison test (a, b) or Dunett’s post hoc analysis (d-j). ****P ≤ 0.0001 siNT = non-targeting siRNA; siSAA1 = SAA1-targeting siRNA. ns=not significant.

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Western Blot, Two Tailed Test, Comparison

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: Basal (a) SAA1 and (b) FPR2 expression in bronchial epithelial from asthmatic patients and matched controls. Data represents means ± SEM of (a and b) n= 6 control and n= 6 asthmatic patients per group. x.

Article Snippet: For in vivo SAA1 neutralization, wild-type BALB/cJ mice were exposed i.t. to PBS, HDM (100 μg), or HDM in combination with a goat anti-mouse SAA1 antibody (R&D Systems, 5 μg/mouse) were sacrificed as indicated.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Promoting mechanism of serum amyloid a family expression in mouse intestinal epithelial cells

doi: 10.1371/journal.pone.0264836

Figure Lengend Snippet: (A) Normalized CRP and SAA expression levels in the liver and each part of the intestinal tract 7 days after DSS administration. (B) After DSS administration, SAA1 expression level increased in the liver, terminal ileum, ascending colon, and rectum when compared to untreated controls. (C) SAA2 was significantly upregulated in organs other than the ascending colon. (D) SAA3 expression level was significantly increased in the liver and rectum but not in the terminal ileum and ascending colon. In particular, SAA3 expression level was strongly increased in the rectum. (E) SAA4 expression was significantly upregulated in the liver and rectum. On the contrary, in the ascending colon, treatment with DSS suppressed the expression of SAA4 . Data are represented as the mean ± standard deviation (n = 6) upon normalization to ACTB expression. **P < 0.01 and *P < 0.05 versus untreated controls. N.D., not detected; L, liver; TI, terminal ileum; PC, proximal colon; R, rectum.

Article Snippet: Recombinant mouse IL-23 (#1877-ML) and anti-SAA1 goat antibody (#AF2948) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Promoting mechanism of serum amyloid a family expression in mouse intestinal epithelial cells

doi: 10.1371/journal.pone.0264836

Figure Lengend Snippet: Normalized SAA expression levels in small intestinal organoids upon stimulation with (A) IL-6 (10 ng/mL), (B) TNF-α (10 ng/mL), (C) IL-1β (100 ng/mL), (D) IL-23 (50 ng/mL), (E) IL-22 (50 ng/mL), or (F) IL-10 (50 ng/mL) for 24 h. Only IL-6, TNF-α, and IL-22 promoted a significant increase in SAA1 expression level when compared with untreated controls. Although not statistically significant, the expression level of SAA1 tended to increase upon IL-1β stimulation and was suppressed by stimulation with IL-10. It was difficult to evaluate SAA2 and SAA4 expression because their expression levels were not detectable (A-F). The expression level of SAA3 was significantly increased by IL-22 and IL-23 (D, E). However, unexpectedly, SAA3 was downregulated by IL-1β (C). SAA expression level was normalized to that of ACTB and is represented as the mean ± standard deviation (n = 5). **P < 0.01 versus untreated controls. N.S., not significant; N.D., not detected.

Article Snippet: Recombinant mouse IL-23 (#1877-ML) and anti-SAA1 goat antibody (#AF2948) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Promoting mechanism of serum amyloid a family expression in mouse intestinal epithelial cells

doi: 10.1371/journal.pone.0264836

Figure Lengend Snippet: (A) Normalized SAA expression levels in small intestinal organoids upon stimulation with Toll-like receptor ligands flagellin (10 μg/mL) for 3 h. The addition of flagellin significantly increased the expression level of SAA1/3 , an effect that was inhibited by NF-κB inhibitor BAY11-7082 (20 μM). SAA expression level was normalized to that of ACTB and is represented as the mean ± standard deviation (n = 5). **P < 0.01. (B) Immunostaining of mouse small intestinal organoids confirmed that flagellin enhanced SAA1 levels. This effect was suppressed in the presence an NF-κB inhibitor. All samples were photographed under the same conditions. Negative controls were also photographed under the same conditions (exposure time, etc.) to confirm the absence of autofluorescence. Scale bar, 10 μm.

Article Snippet: Recombinant mouse IL-23 (#1877-ML) and anti-SAA1 goat antibody (#AF2948) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Standard Deviation, Immunostaining

Journal: PLoS ONE

Article Title: Promoting mechanism of serum amyloid a family expression in mouse intestinal epithelial cells

doi: 10.1371/journal.pone.0264836

Figure Lengend Snippet: ( A ) Normalized SAA expression levels in small intestinal organoids upon stimulation with flagellin alone (10 μg/mL) or plus 5-ASA (40 mM) after 3 h. SAA1/3 was suppressed by the addition of 5-ASA. SAA expression level was normalized to that of ACTB and is represented as the mean ± standard deviation (n = 5). **P < 0.01, *P < 0.05. ( B ) Small intestinal organoids were stimulated with flagellin at 30 min and 3 h with and without 5-ASA. The protein expression levels of phosphorylated-p65 and IκB were examined by SimpleWestern TM . The protein degradation of IκB was observed after 30 min of flagellin stimulation of small intestinal organoids, and the protein expression of IκBα was also repressed by 5-ASA. The inhibition of p65 phosphorylation by 5-ASA was not apparent at 30 min but clear 3 h after flagellin stimulation. This experiment was performed twice independently, and similar results were obtained. A scan of the original untrimmed gel image is displayed in . ( C ) Flagellin promotes activation of the NF-κB pathway, as observed by immunostaining data showing translocation of NF-κB into the nucleus. Immunofluorescence confirmed that NF-κB translocation into the nucleus was inhibited with the addition of 5-ASA. Each negative control was photographed under the same conditions (exposure time, etc.) to confirm the absence of autofluorescence. Scale bar: 10 μm.

Article Snippet: Recombinant mouse IL-23 (#1877-ML) and anti-SAA1 goat antibody (#AF2948) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Standard Deviation, Inhibition, Phospho-proteomics, Activation Assay, Immunostaining, Translocation Assay, Immunofluorescence, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased IL-17-expressing γδT cells in SAA1 TG mice. A , left , flow cytometry detection of lymph node IL-17A + γδ T cell receptor (TCR) + cells, ( B , left ) IL-17A + CD4 + (Th17) cells, and ( C , left ) lineage − CD45 + CD90.2 + IL-17A + (ILC3) cells, represented by dot blots. Data are representative of quadruplicate experiments. ( A – C , right , upper ) The percentages of IL-17A + γδ TCR + , Th17, and ILC3 cells in 5000 lymph node cells (P1) are shown (n = 4). ( A – C , right , bottom ) The absolute numbers of IL-17A + γδ TCR + , Th17, and ILC3 cells per 5000 lymph node cells (P1) are shown (n = 4). Detailed gating strategies and information for P1 are shown ( Fig. S1 ). Data are expressed as the mean ± standard deviation, and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 and ∗∗ p < 0.01 compared with WT mice.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased IL-17-expressing neutrophils in SAA1 TG mice. A , upper , circulating G-CSF levels measured by western blot using equal volumes (1 μl) of serum. The molecular weight of G-CSF is 19 kDa ( A , bottom ). The ponceau S staining to show that the same volume of serum was loaded is shown ( Fig. S2 ). Densitometry analysis of western blots showing G-CSF quantities, n = 3. B , left , blood CD11b + Ly6G + cells (neutrophils) detected by flow cytometry are represented by a dot blot, n = 4. B , right , the percentages of CD11b + Ly6G + cells and absolute numbers of CD11b + Ly6G + cells per 5000 blood cells (P1) are shown (n = 4). C , left , CD11b + Ly6G + cells gated to IL-17A + Ly6G + cells (IL-17-producing neutrophils) by flow cytometry are represented by a dot blot (n = 4). C , right , the percentage of IL-17A + CD11b + Ly6G + cells and absolute numbers of cells per 5000 blood cells (P1) are shown (n = 4). Detailed gating strategies and information for P1 are shown ( Fig. S3 ). Data are expressed as the mean ± standard deviation, and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 compared with WT mice. The red arrow indicates that A is gated to B . MW, molecular weight.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Molecular Weight, Staining, Flow Cytometry, Dot Blot, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased osteoclast-related gene expression and IL-17 and IL-23 expression in whole bone lysates from 6-month-old SAA1-overexpressing TG mice. A , upper , circulating SAA1 levels measured by western blot using equal volumes (1 μl) of serum from 6-month-old mice. The molecular weight of SAA1 is 12 kDa ( A , bottom ). Densitometry analysis of western blots showing SAA1 quantities, n = 3. The ponceau S staining to show that the same volume of serum was loaded is shown ( Fig. S2 ). Relative mRNA expression levels of ( B ) Ctsk , Tnfsf11 , Nfatc1 , and Acp5 ( C ) Tnfrsf11b , Alpl , Bglap , and Runx2 ( D ) Axin1 , Ctnnb1 , and Ccn4 , and ( E ) Il17a and Il23a , normalized to glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) mRNA expression levels, and relative to WT mice in whole bone lysates. For B–E ; WT; n = 4 and TG; n = 8. Data are expressed as the mean ± standard deviation, and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 and ∗∗ p < 0.01 compared with WT mice. MW; molecular weight.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Gene Expression, Expressing, Western Blot, Molecular Weight, Staining, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased Tnfsf11 mRNA expression levels in bone marrow from 6-month-old SAA1-overexpressing TG mice. A , relative mRNA expression levels of Tnfsf11 normalized to glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) mRNA expression levels and relative to WT mice in bone lysates. B , Tnfsf11/Gapdh ΔCT was calculated by subtracting the CT value for mRNA Gapdh expression levels from the CT value for Tnfsf11 mRNA expression levels. Data are expressed as the mean ± standard deviation, and unpaired two-tailed Student's t -test was applied to compare two groups. For A and B ; WT; n = 4, TG; n = 5. N.D; not detected.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased populations of RANKL-expressing cells in bone marrow from SAA1-overexpressing TG mice. A , left , Total RANKL + cells and ( B , left ) CD45 + RANKL + and CD45 − RANKL + cells in bone marrow were detected by flow cytometry and represented by a dot blot (n = 4). Representative data from quadruplicate experiments are shown. The percentage and absolute numbers of ( A , right ) RANKL + cells and ( B , middle ) CD45 + RANKL + and CD45 − RANKL + cells per 5000 of bone marrow cells (P1) are shown (n = 4). B , right , detailed gating strategies and information for P1 are shown ( Fig. S4 ). Data are expressed as the mean ± standard deviation and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 and ∗∗ p < 0.01 compared with WT mice.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Dot Blot, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased RANKL-expressing innate immune cells in the bone marrow from 6-month-old SAA1 TG mice. A , left , γδ TCR + cells and ( B , left ) CD11b + Ly6G + cells in bone marrow were detected by flow cytometry and represented by dot blots (n = 4). Representative data from quadruplicate experiments are shown. The percentages and absolute numbers of ( A , right ) γδ TCR + cells and ( B , right ) CD11b + Ly6G + cells per 10,000 bone marrow cells (P1) are shown. Detailed gating strategies and information for P1 are shown ( Fig. S5 ). Data are expressed as the mean ± standard deviation and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 and ∗∗ p < 0.01 compared with WT mice.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased RANKL-expressing neutrophils in bone marrow from SAA1 TG mice. A , left , RANKL + γδTCR + cells and ( B , left ) RANKL + CD11b + Ly6G + cells in bone marrow were detected by flow cytometry and represented by dot blots (n = 4). Representative data from quadruplicate experiments are shown. A , right , upper , the percentage of RANKL + γδTCR + cells and ( A , right , bottom ), the counts for RANKL + γδTCR + cells in 10,000 bone marrow cells (P1) are shown (n = 4). B , right , upper , the percentage of RANKL + cells and ( B , right , bottom ), the counts for RANKL + cells per 5000 CD11b + Ly6G + cells in bone marrow (E1) are shown (n = 4). Detailed gating strategies and information for P1 and E1 are shown ( Fig. S6 ). Data are expressed as the mean ± standard deviation and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 compared with WT mice.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Changes in bone mineral density and architecture in SAA1 TG mice. A , upper , bone mineral density, bone volume/total volume, trabecular thickness, trabecular number, and cortical thickness of SAA1 TG and WT mice (n = 8). B , a representative TRAP stained section from a quadruplicate experiment is shown. The red arrows indicate TRAP-positive areas (n = 4). (WT, TG, left ) scale bar = 500 μm, (WT, TG, middle , right ) scale bar = 100 μm. Data are expressed as the mean ± standard deviation and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 and ∗∗ p < 0.01 compared with WT mice.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Staining, Standard Deviation, Two Tailed Test