Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: IFN-φ neutralization abolishes ST-induced interferon production in zebrafish embryos. (A) Representative immunohistochemical staining of IFN-φ (brown DAB precipitate) in 1 dpf wild-type (WT) embryos under four conditions: (a.1) unstimulated control, (a.2) ST stimulation (10 6 cells/ml for 2h), (a.3) anti-IFN-β antibody pretreatment alone, and (a.4) anti-IFN-β pretreatment followed by ST stimulation. Scale bar: 50 μm. (B) Quantification of IFN-φ-positive cells from two slides per group (eight sections/slide, three embryos/section) using Fiji-ImageJ. Data represent mean ± SEM ( n = 3 independent experiments); * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Neutralization, Immunohistochemical staining, Staining, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: IFN-I signaling is essential for IL-1β–mediated protection against ST stimulation. (A) Locomotor activity (total distance traveled) assessed 72h after stimulation at 28°C. Mortality count (C) and Caspy2 expression in WT embryos after ST stimulation (10 6 cells/ml, 2h) with or without anti-IFN-β antibody pretreatment. The corresponding bar graph (D) represents the protein expression levels as a percentage of the control. (E) Quantification and (F) representative IHC images of mature IL-1β (mIL-1β)–positive cells (brown DAB precipitate) in: (f.1) ST-stimulated embryos, (f.2) pretreatment with anti-IFN-β alone, and (f.3) pretreatment with anti-IFN-β followed by ST stimulation. Scale bar: 200 μm. Data represent mean ± SEM ( n = 3 independent experiments); * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Activity Assay, Expressing, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Effectiveness of CRISPR/Cas9 depletion of loc795232. Basal expression of loc795232 mRNA transcripts in unstimulated WT or KO embryos ( n = 100/group) at 1 dpf by RT-qPCR (A) or by in situ hybridization (B) . All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. Data represent mean ± SEM of three biological replicates. Independent groups of 1 dpf embryos previously treated or not with Pam3CSK4 or MCC950 and subjected to ST stimulation for 2h, as well as KO larvae stimulated with ST were processed for Natterin detection by WB (C) using anti-Natterin serum (62 kDa dimeric form) and anti-IgG TrueBlot HRP. The corresponding bar graph represents the protein expression levels as a percentage of the control (D) , and Ponceau S staining is visualized in the first horizontal line.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: CRISPR, Expressing, Quantitative RT-PCR, In Situ Hybridization, Control, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Natterin is required for Gbp4 induction. Constitutive expression of gbp4 (A) and gbp1 (C) , was analyzed in unstimulated WT embryos ( n = 100/group) at 24h intervals by RT-qPCR. 1 dpf ST-responsive expression of gbp4 (B) and gbp1 (D) was assessed in WT and KO groups. All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. * p < 0.05 versus unstimulated control WT; # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Natterin is essential for proteolytic activation of Caspy and Caspy2 during ST stimulation. (A) Developmental expression profile of caspy2 mRNA in unstimulated WT embryos ( n = 100/group) from 24 to 120 hpf. (B) One day post-fertilization ST-induced caspy2 expression in WT versus natterin knockout (KO) embryos 2h post-stimulation. All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. Data represent mean ± SEM; * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT. Western blot analysis of mature Caspy ( Supplementary Figure S3 ) and Caspy2 ( Supplementary Figure S4 ) expression in whole embryo lysates at indicated times post-ST stimulation (15 and 30 min, 1 and 2h). Data are normalized to the unstimulated control and presented as percentage values. Quantitative densitometry is shown in bar graphs (C, D) .

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Activation Assay, Expressing, Knock-Out, Control, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: IFN-φ neutralization abolishes ST-induced interferon production in zebrafish embryos. (A) Representative immunohistochemical staining of IFN-φ (brown DAB precipitate) in 1 dpf wild-type (WT) embryos under four conditions: (a.1) unstimulated control, (a.2) ST stimulation (10 6 cells/ml for 2h), (a.3) anti-IFN-β antibody pretreatment alone, and (a.4) anti-IFN-β pretreatment followed by ST stimulation. Scale bar: 50 μm. (B) Quantification of IFN-φ-positive cells from two slides per group (eight sections/slide, three embryos/section) using Fiji-ImageJ. Data represent mean ± SEM ( n = 3 independent experiments); * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Neutralization, Immunohistochemical staining, Staining, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: IFN-I signaling is essential for IL-1β–mediated protection against ST stimulation. (A) Locomotor activity (total distance traveled) assessed 72h after stimulation at 28°C. Mortality count (C) and Caspy2 expression in WT embryos after ST stimulation (10 6 cells/ml, 2h) with or without anti-IFN-β antibody pretreatment. The corresponding bar graph (D) represents the protein expression levels as a percentage of the control. (E) Quantification and (F) representative IHC images of mature IL-1β (mIL-1β)–positive cells (brown DAB precipitate) in: (f.1) ST-stimulated embryos, (f.2) pretreatment with anti-IFN-β alone, and (f.3) pretreatment with anti-IFN-β followed by ST stimulation. Scale bar: 200 μm. Data represent mean ± SEM ( n = 3 independent experiments); * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Activity Assay, Expressing, Control

Journal: Frontiers in Plant Science

Article Title: Time-Resolved Investigation of Molecular Components Involved in the Induction of NO 3 – High Affinity Transport System in Maize Roots

doi: 10.3389/fpls.2016.01657

Figure Lengend Snippet: Western blot analysis carried out on the microsomal fraction extracted from the root tissue of maize seedlings treated with NO 3 – for the indicated times. (A) Western blot analysis performed using anti-PM H + -ATPase (upper panel), anti-NRT2.1 (middle panel) or anti-NRT3.1A (lower panel) antibodies. The same amount of proteins was loaded in each lane. The protein molecular weights were estimated using the ECL TM Plex Fluorescent Rainbow Markers (GE Healthcare Life Sciences). (B) Densitometric analysis of PM H + -ATPase, NRT2.1, and NRT3.1A. Data are expressed as ratio of the corresponding 0 h sample (signals were quantified using Quantity One software, Bio-Rad). Data are the means ± SE, n = 3.

Article Snippet: The resulting mixture was treated at 37°C for 20 min. For the analysis of NRT2 and NRT3, a second buffer was used (0.05 M Tris-HCl, pH 6.8, 4% (w/v) SDS, 12% (w/v) glycerol, 2% (v/v) 2- mercaptoethanol, and 0.05% (w/v) bromophenol blue), and samples were boiled for 5 min. Five micrograms of ECL TM Plex Fluorescent Rainbow Markers (GE Healthcare Amersham TM ) were used for each SDS-PAGE analysis.

Techniques: Western Blot, Software

Journal: Frontiers in Plant Science

Article Title: Time-Resolved Investigation of Molecular Components Involved in the Induction of NO 3 – High Affinity Transport System in Maize Roots

doi: 10.3389/fpls.2016.01657

Figure Lengend Snippet: Separation of PM H + -ATPase complexes by non-denaturing Deriphat-PAGE followed by SDS-PAGE. (A) Western blot analysis performed using antibodies anti-PM H + -ATPase. The molecular weight of each protein marker (ECL Plex Fluorescent Rainbow Markers) was reported on the right side. (B) Densitometric analysis of PM H + -ATPase. Data are expressed as ratio of corresponding 0 h sample considering the sum of all three forms (signals were quantified using PDQuest TM 2-D Analysis Software, Bio-Rad). Data are the means ± SE, n = 3.

Article Snippet: The resulting mixture was treated at 37°C for 20 min. For the analysis of NRT2 and NRT3, a second buffer was used (0.05 M Tris-HCl, pH 6.8, 4% (w/v) SDS, 12% (w/v) glycerol, 2% (v/v) 2- mercaptoethanol, and 0.05% (w/v) bromophenol blue), and samples were boiled for 5 min. Five micrograms of ECL TM Plex Fluorescent Rainbow Markers (GE Healthcare Amersham TM ) were used for each SDS-PAGE analysis.

Techniques: SDS Page, Western Blot, Molecular Weight, Marker, Software

Journal: Vaccines

Article Title: Vaccination with Ectoparasite Proteins Involved in Midgut Function and Blood Digestion Reduces Salmon Louse Infestations

doi: 10.3390/vaccines8010032

Figure Lengend Snippet: Antibody response in vaccinated fish. ( A ) The recombinant proteins were produced in E. coli and analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blot (WB). Ten micrograms of recombinant proteins was loaded onto a 12% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue or transferred to a nitrocellulose membrane. For Western blot analysis, pooled sera collected from vaccinated salmons before salmon louse infestation at approximately week 10 post-initial vaccination were used as primary antibodies. The membrane was then incubated with mouse anti-rainbow trout antibodies and revealed with a goat anti-mouse IgG-HPR conjugate. ( B ) Antibody titers were determined in vaccinated and control fish by ELISA. Values of D.D. at 450 nm in vaccinated and control groups were compared by Student’s t -test with Welch’s correction for unequal variances ( p = 0.05).

Article Snippet: Plates were washed three times with PBS/0.05% Tween 20, and 100 μL/well of mouse anti-rainbow trout antibodies (Bio-Rad Laboratories, Inc.), diluted (1:1000, v / v ) in blocking solution, was added and incubated for 1 h at RT.

Techniques: Recombinant, Produced, Polyacrylamide Gel Electrophoresis, Western Blot, Staining, Membrane, Incubation, Control, Enzyme-linked Immunosorbent Assay