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fitc anti mouse c3b  (Cedarlane)


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    Cedarlane fitc anti mouse c3b
    Fitc Anti Mouse C3b, supplied by Cedarlane, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/fitc+rat+igg2a/Anti-Mouse+Complement+Component+C3%2C+FITC+(Clone+RmC11H9)+(rat+IgG2a)/pm41416942-258-4-8
    Average 93 stars, based on 19 article reviews
    fitc anti mouse c3b - by Bioz Stars, 2026-11
    93/100 stars

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    Related Articles

    Staining:

    Article Title: Prostaglandin D 2 amplifies lupus disease through basophil accumulation in lymphoid organs
    Article Snippet: They were blocked with PBS containing 5% goat serum (Sigma-Aldrich) for 1 h at room temperature, then washed three times with PBS containing 1% BSA and 0.1% Tween 20. .. Staining was achieved after 2 h of incubation with 5 μg/mL AF488 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc) or FITC anti-mouse C3 (CEDARLANE) or the corresponding isotype controls: AF488-goat IgG (Jackson ImmunoResearch Laboratories, Inc), FITC Rat IgG2a (CEDARLANE), respectively. .. Slides were then mounted in Immuno-mount (Thermo Scientific) and kept overnight at 4 °C.

    Article Title: Basophils contribute to pristane-induced Lupus-like nephritis model.
    Article Snippet: They were blocked with PBS containing 5% goat serum (Sigma-Aldrich) for 1 hour at room temperature, then washed 3 times with PBS containing 1% BSA and 0.1% Tween 20. .. Staining was achieved after 2 h of incubation with 5 μg/mL AF488 goat anti-mouse IgG (Jackson Immunoresearch laboratories, Inc) or FITC anti-mouse C3 (CEDARLANE) or the corresponding isotype controls: AF488-goat IgG (Jackson Immunoresearch laboratories, Inc), FITC Rat IgG2a (CEDARLANE), respectively. .. Slides were then mounted in Immuno-mount (Thermo Scientific) and kept overnight at 4 °C.

    Article Title: Basophils contribute to pristane-induced Lupus-like nephritis model
    Article Snippet: They were blocked with PBS containing 5% goat serum (Sigma-Aldrich) for 1 hour at room temperature, then washed 3 times with PBS containing 1% BSA and 0.1% Tween 20. .. Staining was achieved after 2 h of incubation with 5 μg/mL AF488 goat anti-mouse IgG (Jackson Immunoresearch laboratories, Inc) or FITC anti-mouse C3 (CEDARLANE) or the corresponding isotype controls: AF488-goat IgG (Jackson Immunoresearch laboratories, Inc), FITC Rat IgG2a (CEDARLANE), respectively. .. Slides were then mounted in Immuno-mount (Thermo Scientific) and kept overnight at 4 °C.

    Article Title: Prostaglandin D 2 amplifies lupus disease through basophil accumulation in lymphoid organs.
    Article Snippet: They were blocked with PBS containing 5% goat serum (Sigma-Aldrich) for 1 h at room temperature, then washed three times with PBS containing 1% BSA and 0.1% Tween 20. .. Staining was achieved after 2 h of incubation with 5 μg/mL AF488 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc) or FITC anti-mouse C3 (CEDARLANE) or NATURE COMMUNICATIONS | (2018) 9:725 |DOI: 10.1038/s41467-018-03129-8 |www.nature.com/naturecommunications 11 the corresponding isotype controls: AF488-goat IgG (Jackson ImmunoResearch Laboratories, Inc), FITC Rat IgG2a (CEDARLANE), respectively. .. Slides were then mounted in Immuno-mount (Thermo Scientific) and kept overnight at 4 °C.

    Incubation:

    Article Title: Prostaglandin D 2 amplifies lupus disease through basophil accumulation in lymphoid organs
    Article Snippet: They were blocked with PBS containing 5% goat serum (Sigma-Aldrich) for 1 h at room temperature, then washed three times with PBS containing 1% BSA and 0.1% Tween 20. .. Staining was achieved after 2 h of incubation with 5 μg/mL AF488 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc) or FITC anti-mouse C3 (CEDARLANE) or the corresponding isotype controls: AF488-goat IgG (Jackson ImmunoResearch Laboratories, Inc), FITC Rat IgG2a (CEDARLANE), respectively. .. Slides were then mounted in Immuno-mount (Thermo Scientific) and kept overnight at 4 °C.

    Article Title: Basophils contribute to pristane-induced Lupus-like nephritis model.
    Article Snippet: They were blocked with PBS containing 5% goat serum (Sigma-Aldrich) for 1 hour at room temperature, then washed 3 times with PBS containing 1% BSA and 0.1% Tween 20. .. Staining was achieved after 2 h of incubation with 5 μg/mL AF488 goat anti-mouse IgG (Jackson Immunoresearch laboratories, Inc) or FITC anti-mouse C3 (CEDARLANE) or the corresponding isotype controls: AF488-goat IgG (Jackson Immunoresearch laboratories, Inc), FITC Rat IgG2a (CEDARLANE), respectively. .. Slides were then mounted in Immuno-mount (Thermo Scientific) and kept overnight at 4 °C.

    Article Title: Basophils contribute to pristane-induced Lupus-like nephritis model
    Article Snippet: They were blocked with PBS containing 5% goat serum (Sigma-Aldrich) for 1 hour at room temperature, then washed 3 times with PBS containing 1% BSA and 0.1% Tween 20. .. Staining was achieved after 2 h of incubation with 5 μg/mL AF488 goat anti-mouse IgG (Jackson Immunoresearch laboratories, Inc) or FITC anti-mouse C3 (CEDARLANE) or the corresponding isotype controls: AF488-goat IgG (Jackson Immunoresearch laboratories, Inc), FITC Rat IgG2a (CEDARLANE), respectively. .. Slides were then mounted in Immuno-mount (Thermo Scientific) and kept overnight at 4 °C.

    Article Title: Prostaglandin D 2 amplifies lupus disease through basophil accumulation in lymphoid organs.
    Article Snippet: They were blocked with PBS containing 5% goat serum (Sigma-Aldrich) for 1 h at room temperature, then washed three times with PBS containing 1% BSA and 0.1% Tween 20. .. Staining was achieved after 2 h of incubation with 5 μg/mL AF488 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc) or FITC anti-mouse C3 (CEDARLANE) or NATURE COMMUNICATIONS | (2018) 9:725 |DOI: 10.1038/s41467-018-03129-8 |www.nature.com/naturecommunications 11 the corresponding isotype controls: AF488-goat IgG (Jackson ImmunoResearch Laboratories, Inc), FITC Rat IgG2a (CEDARLANE), respectively. .. Slides were then mounted in Immuno-mount (Thermo Scientific) and kept overnight at 4 °C.

    other:

    Article Title: Protein tyrosine phosphatase 1B (PTP1B) is dispensable for IgE-mediated cutaneous reaction in vivo.
    Article Snippet: Contents lists available at ScienceDirect Cellular Immunology journal homepage: www.elsevier .com/locate /yc imm Research paper Protein tyrosine phosphatase 1B (PTP1B) is dispensable for IgE-mediated cutaneous reaction in vivo http://dx.doi.org/10.1016/j.cellimm.2016.05.005 0008-8749/ 2016 Elsevier Inc. Abbreviations: BMMC, bone marrow-derived mast cell; BSA, bovine serum albumin; FBS, fetal bovine serum; TNP, trinitrophenyl; DNFB, dinitroflurobenzene; DNP, dinitrophenyl; PTP1B, protein tyrosine phosphatase 1B; STAT5, signal transducer and activator of transcription 5; OVA, ovalbumin; SCF, stem cell factor; HBSS, Hank’s Balanced Salt Solution; PGE2, prostaglandin E2; IjB, inhibitor of NFjB; AKT, protein kinase B; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; IL, interleukin; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; NFAT, nuclear factor of activated T cells; TNF, tumour necrosis factor; JAK, Janus kinase; TYK, tyrosine kinase; RTK, receptor tyrosine kinase.. ⇑ Corresponding author at: The Institute of Medical Biology, Chinese Academy of Medical Sciences, 935 Jiaoling Road, Kunming, Yunnan 650118, China.

    Article Title: Rcan1 negatively regulates FcεRI-mediated signaling and mast cell function
    Article Snippet: Antibodies to p38 MAPK and actin were purchased from Santa Cruz Biotechnology, Inc. FITC-conjugated rat anti–mouse CD117 (c-kit) mAb and FITC–rat IgG2a were purchased from Cedarlane Laboratories Ltd. FITC-conjugated rat anti–mouse IgE (IgG1) and FITC–rat IgG1 were purchased from BD.

    Article Title: Regulator of Calcineurin 1 (Rcan1) Is Required for the Development of Pulmonary Eosinophilia in Allergic Inflammation in Mice
    Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD117 (c-kit) monoclonal antibody and FITC-rat IgG2a were purchased from Cedarlane Laboratories (Hornby, ON, Canada).



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    (A.) Immunofluorescence staining of γ-Η2A.X and 53BP1 in wt untreated, wt LPS-treated (wt LPS ) and Er1 Lyz2/− BMDMs. Cells with over 2 colocalized foci of the two proteins were labeled positive and indicated using white arrows. The percentage of positive cells is plotted. (At least 4 independent optical fields were counted from n = 3 biological replicates.) Single-channel images of Extended Data Fig. 7A are shown in Supplementary file ( B.) Flow cytometry analysis of the MHC-II expression levels in wt untreated, wt LPS and Er1 Lyz2/− BMDMs. Representative histograms and MFIs are plotted (n = 3-5). (C.) Immunoprecipitation and western blot detection of the MHC-II protein in BMDMs using an anti-MHC-II (IP) or isotype control antibody <t>(IgG).</t> (D.) Volcano plot of differentially presented peptides in wt (downregulated, blue) and wt LPS cells (upregulated, red). Log 2 (Fold Change) of -6 or 6 represents peptides uniquely identified in the wt or wt LPS MHC-peptidome. Statistical significance (ANOVA analysis) was set at p-value ≤ 0.05 (horizontal black dashed line) and peptide enrichment at Log 2 (Fold Change)≥0.3 (upregulated in wt LPS ) or Log 2 (Fold Change)≤-0.3 (downregulated in wt LPS ) (vertical black dashed line). Extracellular matrix proteins are labeled with their corresponding gene symbol. (E.) Bubble plot of the Gene Ontology (GO) term enrichment analysis (cellular component, Mann-Whitney U test) of significantly over-represented wt LPS peptides, when compared to wt controls (p-value ≤ 0.05 and Log 2 Fold change≥0.3). The dot size shows the total count of genes per annotated pathway and the blue color scale indicates the statistical significance as per the p-value of the enriched pathways. The x axis indicates the fold enrichment derived from pathway analysis. (F.) IFN-γ ELISpot analysis of splenocytes isolated from either 8-month-old wt or Er1 Lyz2/− mice and pulsed with the indicated peptides. Representative images are shown. Error bars indicate S.E.M. among replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01 (two-tailed Student’s t-test). Scale bars: 10μm.
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    Podocyte damage and inflammatory changes are worsened by HPD in systemic GC-A knockout mice. ( a ) Immunohistochemical (IHC) staining for WT-1 in glomerulus. The graph shows the number of WT-1-positive cells per glomerulus as quantitative analysis. ( b ) IF staining for nephrin in each group. Scale Bar represents 50 μm. ( c ) Representative images of electron microscopic analysis in each group. The graphs indicate quantitative analysis of foot process width and GBM thickness. Scale Bar represents 1 μm. ( d ) IHC staining of <t>MAC2</t> in glomerulus (▶). The graph shows the number of MAC2-positive macrophages per glomerulus. Scale Bar represents 50 μm. ( e , f ) Glomerular mRNA expression levels of Tgfb1 , Ctgf , Serpine1 , Ccl2 , Emr1 , Icam1 , and Vcam1 in each group. ( g ) Renal mRNA expression levels of Agt , Ren1 , and Sgk1 in each group. β-Actin ( Actb ) was used as internal control. Values are expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, NS; not significant.
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    (A.) Immunofluorescence staining of γ-Η2A.X and 53BP1 in wt untreated, wt LPS-treated (wt LPS ) and Er1 Lyz2/− BMDMs. Cells with over 2 colocalized foci of the two proteins were labeled positive and indicated using white arrows. The percentage of positive cells is plotted. (At least 4 independent optical fields were counted from n = 3 biological replicates.) Single-channel images of Extended Data Fig. 7A are shown in Supplementary file ( B.) Flow cytometry analysis of the MHC-II expression levels in wt untreated, wt LPS and Er1 Lyz2/− BMDMs. Representative histograms and MFIs are plotted (n = 3-5). (C.) Immunoprecipitation and western blot detection of the MHC-II protein in BMDMs using an anti-MHC-II (IP) or isotype control antibody (IgG). (D.) Volcano plot of differentially presented peptides in wt (downregulated, blue) and wt LPS cells (upregulated, red). Log 2 (Fold Change) of -6 or 6 represents peptides uniquely identified in the wt or wt LPS MHC-peptidome. Statistical significance (ANOVA analysis) was set at p-value ≤ 0.05 (horizontal black dashed line) and peptide enrichment at Log 2 (Fold Change)≥0.3 (upregulated in wt LPS ) or Log 2 (Fold Change)≤-0.3 (downregulated in wt LPS ) (vertical black dashed line). Extracellular matrix proteins are labeled with their corresponding gene symbol. (E.) Bubble plot of the Gene Ontology (GO) term enrichment analysis (cellular component, Mann-Whitney U test) of significantly over-represented wt LPS peptides, when compared to wt controls (p-value ≤ 0.05 and Log 2 Fold change≥0.3). The dot size shows the total count of genes per annotated pathway and the blue color scale indicates the statistical significance as per the p-value of the enriched pathways. The x axis indicates the fold enrichment derived from pathway analysis. (F.) IFN-γ ELISpot analysis of splenocytes isolated from either 8-month-old wt or Er1 Lyz2/− mice and pulsed with the indicated peptides. Representative images are shown. Error bars indicate S.E.M. among replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01 (two-tailed Student’s t-test). Scale bars: 10μm.

    Journal: Nature Aging

    Article Title: DNA damage in macrophages drives immune autoreactivity via nuclear antigen presentation

    doi: 10.1038/s43587-025-01053-3

    Figure Lengend Snippet: (A.) Immunofluorescence staining of γ-Η2A.X and 53BP1 in wt untreated, wt LPS-treated (wt LPS ) and Er1 Lyz2/− BMDMs. Cells with over 2 colocalized foci of the two proteins were labeled positive and indicated using white arrows. The percentage of positive cells is plotted. (At least 4 independent optical fields were counted from n = 3 biological replicates.) Single-channel images of Extended Data Fig. 7A are shown in Supplementary file ( B.) Flow cytometry analysis of the MHC-II expression levels in wt untreated, wt LPS and Er1 Lyz2/− BMDMs. Representative histograms and MFIs are plotted (n = 3-5). (C.) Immunoprecipitation and western blot detection of the MHC-II protein in BMDMs using an anti-MHC-II (IP) or isotype control antibody (IgG). (D.) Volcano plot of differentially presented peptides in wt (downregulated, blue) and wt LPS cells (upregulated, red). Log 2 (Fold Change) of -6 or 6 represents peptides uniquely identified in the wt or wt LPS MHC-peptidome. Statistical significance (ANOVA analysis) was set at p-value ≤ 0.05 (horizontal black dashed line) and peptide enrichment at Log 2 (Fold Change)≥0.3 (upregulated in wt LPS ) or Log 2 (Fold Change)≤-0.3 (downregulated in wt LPS ) (vertical black dashed line). Extracellular matrix proteins are labeled with their corresponding gene symbol. (E.) Bubble plot of the Gene Ontology (GO) term enrichment analysis (cellular component, Mann-Whitney U test) of significantly over-represented wt LPS peptides, when compared to wt controls (p-value ≤ 0.05 and Log 2 Fold change≥0.3). The dot size shows the total count of genes per annotated pathway and the blue color scale indicates the statistical significance as per the p-value of the enriched pathways. The x axis indicates the fold enrichment derived from pathway analysis. (F.) IFN-γ ELISpot analysis of splenocytes isolated from either 8-month-old wt or Er1 Lyz2/− mice and pulsed with the indicated peptides. Representative images are shown. Error bars indicate S.E.M. among replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01 (two-tailed Student’s t-test). Scale bars: 10μm.

    Article Snippet: Antibodies and isotype controls were purchased from BioLegend and Proteintech: anti-CD19 (BioLegend, 152410; clone 1D3/CD19), anti-CD138 (BioLegend, 142503; clone 281-2), anti-CD11b (BioLegend, 101212; clone M1I70), anti-CD4 (BioLegend, 100406, 100412 and 100432; clone GK1.5), anti-CD25 (BioLegend, 102012; clone PC61), anti-FOXP3 (Proteintech, PE-65089; clone 3G3), anti-MHC-II (BioLegend, 107606, 107636 and 107631; clone M5/114.15.2), anti-CD86 (BioLegend, 105026; clone GL-1), anti-Ly6G (BioLegend, 127654 and 127607; clone 1A8), anti-CD62L (BioLegend, 104412; clone MEL-14), anti-CD44 (BioLegend, 103036; clone IM7), anti-T-bet (BioLegend, 644812; clone 4B10), anti-PD-1 (BioLegend, 135214; clone 29F.1A12), anti-CD69 (BioLegend, 104507; clone H1.2F3), anti-F4/80 (BioLegend, 123110; clone BM8), anti-CD115 (BioLegend, 135512; clone AFS98), anti-mouse Lineage Cocktail with Isotype Ctrl (BioLegend, 133305; clones 17A2, RB6-8C5, RA3-6B2, Ter119 and M1/70), anti-mouse CD34 antibody (BioLegend, 119307; clone MEC14.7), anti-mouse Ly-6A/E (Sca-1) (BioLegend, 108123; clone D7), anti-mouse CD16/32 (BioLegend, 101325; clone 93), anti-mouse c-Kit (BioLegend, 105815; clone 2B8), anti-mouse CD170 (Siglec-F) antibody (BioLegend, 155523; clone S17007L), anti-mouse NK-1.1 (BioLegend, 108705; clone PK316), anti-mouse CD3a (Proteintech, PE-65060), anti-mouse FoxP3 (BioLegend, 126409; clone MF-14), i-mouse CD8a (BioLegend, 100712; clone 53-6.7), PerCP rat IgG2a (BioLegend, 400529; clone RTK2758) and FITC rat IgG2a (Proteintech, FITC-65209; clone 2A3) were used as isotype control antibodies.

    Techniques: Immunofluorescence, Staining, Labeling, Flow Cytometry, Expressing, Immunoprecipitation, Western Blot, Control, MANN-WHITNEY, Derivative Assay, Enzyme-linked Immunospot, Isolation, Two Tailed Test

    (A) Schematic showing the structure of PD-L2 (left) containing the extracellular IgV and IgC domains, the TM (transmembrane) domain and a short cytoplasmic domain. Sequences of the last 7 amino acids of the TM domain and the whole cytoplasmic domain are shown for the mouse C57BL/6J (B6) and CAST/EiJ ( Mus musculus castaneus ) strains, and several other mammals, indicating amino acid numbering and amino acids conserved between multiple mammalian species in green. Mutation of the stop codon in B6 mice to tryptophan (W248) in the corrected mouse B6 strain (X248W) results in a cytoplasmic domain very similar to that in the CAST/EiJ mouse. (B) Graphs of percentage of B-1a, B-1b and B-2 cells among all peritoneal cavity CD19 + B cells (gated as in ) in PD-L2ko, WT and X248W mouse strains (n=10). (C-H) Flow cytometric analysis of peritoneal cavity B-1a cells from the indicated strains, gated as in , showing representative plots for expression of PC1 (C), CD73 (E) and IgG3 (G) and graphs showing percentage of B-1a cells positive for the same three proteins (D, F, H). Gates set using B-2 cells which are largely negative for both proteins . Sample numbers: PC1, PD-L2ko (n=6), WT (n=9) and X248W (n=9); CD73, PD-L2ko (n=5), WT (n=7) and X248W (n=9); IgG3, PD-L2ko (n=8), WT (n=5) and X248W (n=6). (I, J) Flow cytometric analysis of splenocytes from indicated strains showing IgG1 expression on B-1a cells (I). Graph shows percentage of IgG1 + B-1a cells (J). Sample numbers: PD-L2ko (n=7), WT (n6), X248W (n=7). Numbers indicate percentage of cells in gates. Each dot represents one mouse. Horizontal line indicates mean. Data pooled from 3 (B, D, F, H) or 2 (J) independent experiments. Mann-Whitney U test was used for statistical analysis; * 0.01 < p < 0.05 ** 0.001 < p < 0.01, *** 0.0001 < p < 0.001.

    Journal: bioRxiv

    Article Title: PD-L2 regulates natural antibody and IL-10 secretion by B-1 cells

    doi: 10.64898/2025.12.11.693708

    Figure Lengend Snippet: (A) Schematic showing the structure of PD-L2 (left) containing the extracellular IgV and IgC domains, the TM (transmembrane) domain and a short cytoplasmic domain. Sequences of the last 7 amino acids of the TM domain and the whole cytoplasmic domain are shown for the mouse C57BL/6J (B6) and CAST/EiJ ( Mus musculus castaneus ) strains, and several other mammals, indicating amino acid numbering and amino acids conserved between multiple mammalian species in green. Mutation of the stop codon in B6 mice to tryptophan (W248) in the corrected mouse B6 strain (X248W) results in a cytoplasmic domain very similar to that in the CAST/EiJ mouse. (B) Graphs of percentage of B-1a, B-1b and B-2 cells among all peritoneal cavity CD19 + B cells (gated as in ) in PD-L2ko, WT and X248W mouse strains (n=10). (C-H) Flow cytometric analysis of peritoneal cavity B-1a cells from the indicated strains, gated as in , showing representative plots for expression of PC1 (C), CD73 (E) and IgG3 (G) and graphs showing percentage of B-1a cells positive for the same three proteins (D, F, H). Gates set using B-2 cells which are largely negative for both proteins . Sample numbers: PC1, PD-L2ko (n=6), WT (n=9) and X248W (n=9); CD73, PD-L2ko (n=5), WT (n=7) and X248W (n=9); IgG3, PD-L2ko (n=8), WT (n=5) and X248W (n=6). (I, J) Flow cytometric analysis of splenocytes from indicated strains showing IgG1 expression on B-1a cells (I). Graph shows percentage of IgG1 + B-1a cells (J). Sample numbers: PD-L2ko (n=7), WT (n6), X248W (n=7). Numbers indicate percentage of cells in gates. Each dot represents one mouse. Horizontal line indicates mean. Data pooled from 3 (B, D, F, H) or 2 (J) independent experiments. Mann-Whitney U test was used for statistical analysis; * 0.01 < p < 0.05 ** 0.001 < p < 0.01, *** 0.0001 < p < 0.001.

    Article Snippet: Intracellular antibodies used, indicating antigen and fluorophore (clone): Blimp-1 APC (5E7) and IRF4 PE (IRF4.3E4) (both BioLegend), CstF-64 CL488 (rabbit polyclonal) (Proteintech); Isotype controls: Rat IgG2a APC isotype control (G013C12), rat IgG1 PE isotype control (G0114F7) (both BioLegend), rabbit IgG1 AF488 (EPR25A; BD Biosciences).

    Techniques: Mutagenesis, Expressing, MANN-WHITNEY

    (A) Comparison of sequences of the last 7 amino acids of the transmembrane (TM) domain and the whole cytoplasmic domain of PD-L2 for the indicated mouse strains, including C57BL/6J (B6), the corrected C57BL/6J (B6) strain with the X248W mutation and CAST/EiJ ( Mus musculus castaneus ), indicating amino acid numbering and amino acids identical between strains in green. (B) Diagram showing the amino acid sequence of the last 7 amino acids of the TM domain and the reconstituted cytoplasmic domain of PD-L2 X248W with residues conserved between different mammals in green (from ) and numbers showing amino acid positions. Upper panel: predicted phosphorylation sites Y252 and S257 are indicated in red with kinases that may phosphorylate these listed below. Lower panel: predicted binding partners to these two residues and P259 indicated below. Confidence scores of predicted kinases (from NetPhos 3.1) and binding partners (from Scansite 4.0): INSR (0.521), PKA (0.770); ITK (0.580), NEK10 (0.655), SHC1 (0.691), PKA (0.693), NEK2 (0.910), 14-3-3 (0.733), AURKA (0.703), SRC (0.619). (C) Histograms showing PD-L2 expression on B-1a (CD11b + CD5 + ) and B-1b (CD11b + CD5 - ) cells from peritoneal cavity of WT and PD-L2 X248W mice, pre-gated on live CD19 + cells. (D) Graphs of PD-L2 expression on B-1a and B-1b cells (n=10). (E, F) Flow cytometric analysis of PC1 (E) and CD73 (F) expression on peritoneal B-2 cells (CD19 + CD11b - ) showing example flow plots and graphs of frequencies of PC1 + and CD73 + B-2 cells from mice of the indicated genotypes. These gates were used to set gates for PC1 + and CD73 + B-1 cells in . Sample numbers: n=4 for PC1 in PD-L2ko and all CD73 analyses, 5 for PC1 in X248W and 6 for PC1 in WT. (G) Graphs showing frequency of IgG3-secreting B-1 cells (spot forming units, SFU) and amount of IgG3 production (spot size) determined using ELISpot assays on peritoneal B-1 cells from mice of the indicated genotypes incubated for 48 h (n=4 for PD-L2ko and WT, 5 for X248W). (H) Flow cytometric analysis of intracellular Blimp-1 detected with an antibody in B-2, B-1a and B-1b peritoneal cavity cells from a mouse expressing Blimp-1-GFP. Numbers indicate percentage of cells in gates. Each dot represents one mouse. Horizontal line indicates mean. Data pooled from 4 (D) or 2 (E-G) independent experiments. Mann-Whitney U test was used for statistical analysis; * 0.01 < p < 0.05 ** 0.001 < p < 0.01.

    Journal: bioRxiv

    Article Title: PD-L2 regulates natural antibody and IL-10 secretion by B-1 cells

    doi: 10.64898/2025.12.11.693708

    Figure Lengend Snippet: (A) Comparison of sequences of the last 7 amino acids of the transmembrane (TM) domain and the whole cytoplasmic domain of PD-L2 for the indicated mouse strains, including C57BL/6J (B6), the corrected C57BL/6J (B6) strain with the X248W mutation and CAST/EiJ ( Mus musculus castaneus ), indicating amino acid numbering and amino acids identical between strains in green. (B) Diagram showing the amino acid sequence of the last 7 amino acids of the TM domain and the reconstituted cytoplasmic domain of PD-L2 X248W with residues conserved between different mammals in green (from ) and numbers showing amino acid positions. Upper panel: predicted phosphorylation sites Y252 and S257 are indicated in red with kinases that may phosphorylate these listed below. Lower panel: predicted binding partners to these two residues and P259 indicated below. Confidence scores of predicted kinases (from NetPhos 3.1) and binding partners (from Scansite 4.0): INSR (0.521), PKA (0.770); ITK (0.580), NEK10 (0.655), SHC1 (0.691), PKA (0.693), NEK2 (0.910), 14-3-3 (0.733), AURKA (0.703), SRC (0.619). (C) Histograms showing PD-L2 expression on B-1a (CD11b + CD5 + ) and B-1b (CD11b + CD5 - ) cells from peritoneal cavity of WT and PD-L2 X248W mice, pre-gated on live CD19 + cells. (D) Graphs of PD-L2 expression on B-1a and B-1b cells (n=10). (E, F) Flow cytometric analysis of PC1 (E) and CD73 (F) expression on peritoneal B-2 cells (CD19 + CD11b - ) showing example flow plots and graphs of frequencies of PC1 + and CD73 + B-2 cells from mice of the indicated genotypes. These gates were used to set gates for PC1 + and CD73 + B-1 cells in . Sample numbers: n=4 for PC1 in PD-L2ko and all CD73 analyses, 5 for PC1 in X248W and 6 for PC1 in WT. (G) Graphs showing frequency of IgG3-secreting B-1 cells (spot forming units, SFU) and amount of IgG3 production (spot size) determined using ELISpot assays on peritoneal B-1 cells from mice of the indicated genotypes incubated for 48 h (n=4 for PD-L2ko and WT, 5 for X248W). (H) Flow cytometric analysis of intracellular Blimp-1 detected with an antibody in B-2, B-1a and B-1b peritoneal cavity cells from a mouse expressing Blimp-1-GFP. Numbers indicate percentage of cells in gates. Each dot represents one mouse. Horizontal line indicates mean. Data pooled from 4 (D) or 2 (E-G) independent experiments. Mann-Whitney U test was used for statistical analysis; * 0.01 < p < 0.05 ** 0.001 < p < 0.01.

    Article Snippet: Intracellular antibodies used, indicating antigen and fluorophore (clone): Blimp-1 APC (5E7) and IRF4 PE (IRF4.3E4) (both BioLegend), CstF-64 CL488 (rabbit polyclonal) (Proteintech); Isotype controls: Rat IgG2a APC isotype control (G013C12), rat IgG1 PE isotype control (G0114F7) (both BioLegend), rabbit IgG1 AF488 (EPR25A; BD Biosciences).

    Techniques: Comparison, Mutagenesis, Sequencing, Phospho-proteomics, Binding Assay, Expressing, Enzyme-linked Immunospot, Incubation, MANN-WHITNEY

    Podocyte damage and inflammatory changes are worsened by HPD in systemic GC-A knockout mice. ( a ) Immunohistochemical (IHC) staining for WT-1 in glomerulus. The graph shows the number of WT-1-positive cells per glomerulus as quantitative analysis. ( b ) IF staining for nephrin in each group. Scale Bar represents 50 μm. ( c ) Representative images of electron microscopic analysis in each group. The graphs indicate quantitative analysis of foot process width and GBM thickness. Scale Bar represents 1 μm. ( d ) IHC staining of MAC2 in glomerulus (▶). The graph shows the number of MAC2-positive macrophages per glomerulus. Scale Bar represents 50 μm. ( e , f ) Glomerular mRNA expression levels of Tgfb1 , Ctgf , Serpine1 , Ccl2 , Emr1 , Icam1 , and Vcam1 in each group. ( g ) Renal mRNA expression levels of Agt , Ren1 , and Sgk1 in each group. β-Actin ( Actb ) was used as internal control. Values are expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, NS; not significant.

    Journal: Scientific Reports

    Article Title: Finerenone ameliorates diabetic kidney disease exacerbated by deletion of natriuretic peptide/guanylyl cyclase-A signaling and dietary high-protein load

    doi: 10.1038/s41598-025-25362-0

    Figure Lengend Snippet: Podocyte damage and inflammatory changes are worsened by HPD in systemic GC-A knockout mice. ( a ) Immunohistochemical (IHC) staining for WT-1 in glomerulus. The graph shows the number of WT-1-positive cells per glomerulus as quantitative analysis. ( b ) IF staining for nephrin in each group. Scale Bar represents 50 μm. ( c ) Representative images of electron microscopic analysis in each group. The graphs indicate quantitative analysis of foot process width and GBM thickness. Scale Bar represents 1 μm. ( d ) IHC staining of MAC2 in glomerulus (▶). The graph shows the number of MAC2-positive macrophages per glomerulus. Scale Bar represents 50 μm. ( e , f ) Glomerular mRNA expression levels of Tgfb1 , Ctgf , Serpine1 , Ccl2 , Emr1 , Icam1 , and Vcam1 in each group. ( g ) Renal mRNA expression levels of Agt , Ren1 , and Sgk1 in each group. β-Actin ( Actb ) was used as internal control. Values are expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, NS; not significant.

    Article Snippet: Primary antibodies used for immunohistochemical and immunofluorescence studies were anti-GC-A antibody (GTX109810, GeneTex, Inc., Irvine, CA, USA), anti-MAC2 antibody (CL8942F, Cedarlane, Ontario, Canada), anti-Wilms tumor 1 (WT1) antibody (sc-15421, Santa Cruz Biotechnology, Dallas, TX), anti-nephrin antibody (AF3159, R&D Systems, Inc., Minneapolis, MN, USA), and anti-Tie2 antibody (AF762, R&D Systems, Inc.).

    Techniques: Knock-Out, Immunohistochemical staining, Immunohistochemistry, Staining, Expressing, Control

    Podocyte damage and profibrotic changes are worsened by HPD in endothelial cell-specific GC-A knockout mice. ( a ) IHC staining of WT-1 in glomerulus. The graph shows the number of WT-1-positive cells per glomerulus as quantitative analysis. Scale Bar represents 50 μm. ( b ) IF staining for nephrin. Scale Bar represents 50 μm. ( c ) Representative images of electron microscopic analysis in each group. The graphs indicate quantitative analysis of foot process width and GBM thickness. Scale Bar represents 1 μm. ( d ) IHC staining of MAC2 in glomerulus (▶). The graph shows the number of MAC2-positive macrophages per glomerulus. Scale Bar represents 100 μm. ( e ) Glomerular mRNA expression levels of Tgfb1 , Ctgf , Fn1 , Serpine1 , Ccl2 , and Emr1 in each group. ( f ) Renal mRNA levels of Fn1 , Serpine1 , Lcn2 , Agt, and Sgk1 in each group. β-Actin ( Actb ) was used as internal control. Values are expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, NS; not significant.

    Journal: Scientific Reports

    Article Title: Finerenone ameliorates diabetic kidney disease exacerbated by deletion of natriuretic peptide/guanylyl cyclase-A signaling and dietary high-protein load

    doi: 10.1038/s41598-025-25362-0

    Figure Lengend Snippet: Podocyte damage and profibrotic changes are worsened by HPD in endothelial cell-specific GC-A knockout mice. ( a ) IHC staining of WT-1 in glomerulus. The graph shows the number of WT-1-positive cells per glomerulus as quantitative analysis. Scale Bar represents 50 μm. ( b ) IF staining for nephrin. Scale Bar represents 50 μm. ( c ) Representative images of electron microscopic analysis in each group. The graphs indicate quantitative analysis of foot process width and GBM thickness. Scale Bar represents 1 μm. ( d ) IHC staining of MAC2 in glomerulus (▶). The graph shows the number of MAC2-positive macrophages per glomerulus. Scale Bar represents 100 μm. ( e ) Glomerular mRNA expression levels of Tgfb1 , Ctgf , Fn1 , Serpine1 , Ccl2 , and Emr1 in each group. ( f ) Renal mRNA levels of Fn1 , Serpine1 , Lcn2 , Agt, and Sgk1 in each group. β-Actin ( Actb ) was used as internal control. Values are expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, NS; not significant.

    Article Snippet: Primary antibodies used for immunohistochemical and immunofluorescence studies were anti-GC-A antibody (GTX109810, GeneTex, Inc., Irvine, CA, USA), anti-MAC2 antibody (CL8942F, Cedarlane, Ontario, Canada), anti-Wilms tumor 1 (WT1) antibody (sc-15421, Santa Cruz Biotechnology, Dallas, TX), anti-nephrin antibody (AF3159, R&D Systems, Inc., Minneapolis, MN, USA), and anti-Tie2 antibody (AF762, R&D Systems, Inc.).

    Techniques: Knock-Out, Immunohistochemistry, Staining, Expressing, Control