rabbit anti notch1  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: Sex- and time-dependent role of insulin regulated aminopeptidase in lipopolysaccharide-induced inflammation

doi: 10.3389/fimmu.2024.1466692

Figure Lengend Snippet: IRAP gene deletion may result in heightened responsiveness of M1 macrophages in the spleen of male mice 48 hours following LPS treatment. (A) Representative flow cytometry dot plot showing the gating for CD45 + F4/80 + CD206 - M1 macrophages in the spleen, 48-hours following vehicle treatment. (B) The frequency (%) among live CD45 + cells of M1 macrophages in the spleen of male wildtype (WT; filled bars) and IRAP knockout (KO; empty bars) mice (aged 10-15 weeks) administered either vehicle (V; blue) or LPS (L; red) once for 4 hours (n=3) or 24 hours (n=3) or twice over 48 hours (n=6). (C) Quantification of the fold changes in mean fluorescence intensity (MFI) compared to the mean of vehicle controls of the activation markers CD40, CD80, CD86 and MHCII in M1 macrophages. Note that no positive MFI values were measured for CD80 at 4 hours. (D) Frequency curves of MHCII in macrophages from spleens of vehicle- (blue) and LPS-treated (red) WT (filled curves) and IRAP KO (empty curves) mice. Data from each timepoint was analyzed separately using a two-way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01, ***p<0.001. All data is presented as mean ± SEM.

Article Snippet: Antibodies used for immunocytochemistry experiments include BV786-CD45 (BD Bioscience #564225), TNF-α (Abcam #ab6671), IL-1β (Abcam #ab205924), IRAP (Cell Signaling #6918) and TLR4 (Abcam #ab13556).

Techniques: Flow Cytometry, Knock-Out, Fluorescence, Activation Assay

Journal: Frontiers in Immunology

Article Title: Sex- and time-dependent role of insulin regulated aminopeptidase in lipopolysaccharide-induced inflammation

doi: 10.3389/fimmu.2024.1466692

Figure Lengend Snippet: IRAP gene deletion does not appear to influence the LPS-responsiveness of M1 macrophages in the spleen of female mice. (A) Representative flow cytometry dot plot showing the gating for CD45 + F4/80 + CD206 - M1 macrophages in the spleen, 48-hours following vehicle treatment. (B) The frequency (%) among live CD45 + cells of M1 macrophages in the spleen of female wildtype (WT; filled bars) and IRAP knockout (KO; empty bars) mice (aged 10-15 weeks) administered either vehicle (V; blue) or LPS (L; red) once for 4 hours (n=3) or 24 hours (n=3) or twice over 48 hours (n=6). (C) Quantification of the fold changes in mean fluorescence intensity (MFI) compared to the mean of vehicle controls of the activation markers CD40, CD80, CD86 and MHCII in M1 macrophages. (D) Frequency curves of MHCII in macrophages from spleens of vehicle- (blue) and LPS-treated (red) WT (filled curves) and IRAP KO (empty curves) mice. Data from each timepoint was analyzed separately using a two-way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.001. All data is presented as mean ± SEM.

Article Snippet: Antibodies used for immunocytochemistry experiments include BV786-CD45 (BD Bioscience #564225), TNF-α (Abcam #ab6671), IL-1β (Abcam #ab205924), IRAP (Cell Signaling #6918) and TLR4 (Abcam #ab13556).

Techniques: Flow Cytometry, Knock-Out, Fluorescence, Activation Assay

Journal: Frontiers in Immunology

Article Title: Sex- and time-dependent role of insulin regulated aminopeptidase in lipopolysaccharide-induced inflammation

doi: 10.3389/fimmu.2024.1466692

Figure Lengend Snippet: IRAP gene deletion may influence the LPS responsiveness of M2 macrophages in the spleen of male mice. (A) Representative flow cytometry dot plot showing the gating for CD45 + F4/80 + CD206 + M2 macrophages in the spleen, 48-hours following vehicle treatment. (B) The frequency (%) among live CD45 + cells of M2 macrophages in the spleen of male wildtype (WT; filled bars) and IRAP knockout (KO; empty bars) mice (aged 10-15 weeks) administered either vehicle (V; blue) or LPS (L; red) once for 4 hours (n=3) or 24 hours (n=3) or twice over 48 hours (n=6). (C) Quantification of the fold changes in mean fluorescence intensity (MFI) compared to the mean of vehicle controls of the activation markers CD40, CD80, CD86 and MHCII in M2 macrophages. Data from each timepoint was analyzed separately using a two-way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vehicle vs LPS, #p<0.05, ##p<0.01 WT vs KO. All data is presented as mean ± SEM.

Article Snippet: Antibodies used for immunocytochemistry experiments include BV786-CD45 (BD Bioscience #564225), TNF-α (Abcam #ab6671), IL-1β (Abcam #ab205924), IRAP (Cell Signaling #6918) and TLR4 (Abcam #ab13556).

Techniques: Flow Cytometry, Knock-Out, Fluorescence, Activation Assay

Journal: Frontiers in Immunology

Article Title: Sex- and time-dependent role of insulin regulated aminopeptidase in lipopolysaccharide-induced inflammation

doi: 10.3389/fimmu.2024.1466692

Figure Lengend Snippet: IRAP gene deletion does not appear to influence the LPS responsiveness of M2 macrophages in the spleen of female mice. (A) Representative flow cytometry dot plot showing the gating for CD45 + F4/80 + CD206 + M2 macrophages in the spleen, 48-hours following vehicle treatment. (B) The frequency (%) among live CD45 + cells of M2 macrophages in the spleen of female wildtype (WT; filled bars) and IRAP knockout (KO; empty bars) mice (aged 10-15 weeks) administered either vehicle (V; blue) or LPS (L; red) once for 4 hours (n=3) or 24 hours (n=3) or twice over 48 hours (n=6). (C) Quantification of the fold changes in mean fluorescence intensity (MFI) compared to the mean of vehicle controls of the activation markers CD40, CD80, CD86 and MHCII in M2 macrophages. Data from each timepoint was analyzed separately using a two-way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01, ****p<0.0001 vehicle vs LPS, #p<0.05 WT vs KO. All data is presented as mean ± SEM.

Article Snippet: Antibodies used for immunocytochemistry experiments include BV786-CD45 (BD Bioscience #564225), TNF-α (Abcam #ab6671), IL-1β (Abcam #ab205924), IRAP (Cell Signaling #6918) and TLR4 (Abcam #ab13556).

Techniques: Flow Cytometry, Knock-Out, Fluorescence, Activation Assay

Journal: Frontiers in Immunology

Article Title: Sex- and time-dependent role of insulin regulated aminopeptidase in lipopolysaccharide-induced inflammation

doi: 10.3389/fimmu.2024.1466692

Figure Lengend Snippet: IRAP gene deletion results in a heightened pro-inflammatory response in male BMDM. (A) Representative flow cytometry dot plots showing control (C) and LPS-treated (L) CD45 + F4/80 + CD206 - M1 macrophages from male wildtype (WT) mice and (B) the frequencies (%) of the M1 macrophage population in WT (filled bars) and IRAP knockout (KO; empty bars) BMDM cultures. (C) Frequency curves of the activation markers CD40 and CD80 in C (blue) and L (red) WT (filled curve) and IRAP KO (empty curve) M1 macrophages. (D) Quantification of the fold changes in mean fluorescence intensity (MFI) compared to the mean of the controls of CD40, CD80, CD86 and MHCII in M1 macrophages. Data was analyzed using a two-way ANOVA with Tukey’s post-hoc test, **p<0.01 control vs LPS, ##p<0.01 WT vs KO, n=4-5. All data is presented as mean ± SEM.

Article Snippet: Antibodies used for immunocytochemistry experiments include BV786-CD45 (BD Bioscience #564225), TNF-α (Abcam #ab6671), IL-1β (Abcam #ab205924), IRAP (Cell Signaling #6918) and TLR4 (Abcam #ab13556).

Techniques: Flow Cytometry, Control, Knock-Out, Activation Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Sex- and time-dependent role of insulin regulated aminopeptidase in lipopolysaccharide-induced inflammation

doi: 10.3389/fimmu.2024.1466692

Figure Lengend Snippet: IRAP gene deletion does not significantly alter macrophage activation in female BMDM. (A) Representative flow cytometry dot plots showing control (C) and LPS-treated (L) CD45 + F4/80 + CD206 - M1 macrophages from female wildtype (WT) mice and (B) the frequencies (%) of the M1 macrophage population in WT (filled bars) and IRAP knockout (KO; empty bars) BMDM cultures. (C) Frequency curves of the activation markers CD40 and CD80 in C (blue) and L (red) WT (filled curve) and IRAP KO (empty curve) M1 macrophages. (D) Quantification of the fold changes in mean fluorescence intensity (MFI) compared to the mean of the controls of CD40, CD80, CD86 and MHCII in M1 macrophages. Data was analyzed using a two-way ANOVA with Tukey’s post-hoc test, ****p<0.0001 control vs LPS, ##p<0.01, ###p<0.001 WT vs KO, n=3. All data is presented as mean ± SEM.

Article Snippet: Antibodies used for immunocytochemistry experiments include BV786-CD45 (BD Bioscience #564225), TNF-α (Abcam #ab6671), IL-1β (Abcam #ab205924), IRAP (Cell Signaling #6918) and TLR4 (Abcam #ab13556).

Techniques: Activation Assay, Flow Cytometry, Control, Knock-Out, Fluorescence

Journal: Frontiers in Immunology

Article Title: Sex- and time-dependent role of insulin regulated aminopeptidase in lipopolysaccharide-induced inflammation

doi: 10.3389/fimmu.2024.1466692

Figure Lengend Snippet: IRAP-deficient BMDM have heightened LPS-induced increases in TNF-α and IL-1β expression. Representative immunofluorescent staining of (A) TNF-α or (B) IL-1β (green) in control (C) or LPS-treated (L) CD45 + (red) BMDM from male and female wildtype (WT) and IRAP knockout (KO) mice. (C) Protein concentrations (pg/ml) of TNF-α, IL-1β and IL-6 in the conditioned media of BMDM measured using a sandwich ELISA. Data was analyzed using three-way ANOVAs with Tukey’s post-hoc test, **p<0.01, ***p<0.001, n=5-6. Data is presented as mean ± SEM.

Article Snippet: Antibodies used for immunocytochemistry experiments include BV786-CD45 (BD Bioscience #564225), TNF-α (Abcam #ab6671), IL-1β (Abcam #ab205924), IRAP (Cell Signaling #6918) and TLR4 (Abcam #ab13556).

Techniques: Expressing, Staining, Control, Knock-Out, Sandwich ELISA

Journal: Frontiers in Immunology

Article Title: Sex- and time-dependent role of insulin regulated aminopeptidase in lipopolysaccharide-induced inflammation

doi: 10.3389/fimmu.2024.1466692

Figure Lengend Snippet: IRAP expression and distribution changes with LPS stimulation in BMDM. (A) Representative Western blot and (B) densitometric quantification of IRAP (~160 kDa) in cell lysates of untreated control (C; blue) or LPS-treated (L; red) BMDM derived from male and female, wildtype (WT) and IRAP knockout (KO) mice. Data is presented relative to protein concentration and the mean optical density (OD) of the male WT control and was analyzed using a three-way ANOVA with Tukey’s post-hoc test, *p<0.05, n=3 (3 samples per group on separate blots). (C) Representative immunofluorescent images and (D) semi-quantification of % colocalization of IRAP (green) and CD45 (red) in BMDM from WT mice. Data was analyzed using a two-way ANOVA with Tukey’s post-hoc test, n=3. All data is presented as mean ± SEM.

Article Snippet: Antibodies used for immunocytochemistry experiments include BV786-CD45 (BD Bioscience #564225), TNF-α (Abcam #ab6671), IL-1β (Abcam #ab205924), IRAP (Cell Signaling #6918) and TLR4 (Abcam #ab13556).

Techniques: Expressing, Western Blot, Control, Derivative Assay, Knock-Out, Protein Concentration

Journal: Frontiers in Immunology

Article Title: Sex- and time-dependent role of insulin regulated aminopeptidase in lipopolysaccharide-induced inflammation

doi: 10.3389/fimmu.2024.1466692

Figure Lengend Snippet: TLR4 is highly expressed at the cell surface in male IRAP-deficient macrophages. (A) Representative immunofluorescent images of TLR4 (green) in untreated control (C) and LPS-treated (L) CD45 + (red) BMDM derived from male and female wildtype (WT) and IRAP knockout (KO) mice. (B) Semi-quantification of TLR4 expression from immunofluorescent (IF) images of BMDM measured as % area positive staining relative to the cell count. (C) Semi-quantification of the TLR4 expressed at the cell surface of BMDM in IF images measured as % colocalization of TLR4 and CD45. (D) Raw mean fluorescence intensity (MFI) of cell surface TLR4 expression in CD45 + F4/80 + CD206 - M1 macrophages in the spleens of mice treated for 4 hours with vehicle control (V) or LPS (L) measured via flow cytometry. All data was analyzed using three-way ANOVAs with Sidak’s post-hoc test, **p<0.01, n=3. All data is presented as mean ± SEM.

Article Snippet: Antibodies used for immunocytochemistry experiments include BV786-CD45 (BD Bioscience #564225), TNF-α (Abcam #ab6671), IL-1β (Abcam #ab205924), IRAP (Cell Signaling #6918) and TLR4 (Abcam #ab13556).

Techniques: Control, Derivative Assay, Knock-Out, Expressing, Staining, Cell Counting, Fluorescence, Flow Cytometry