igm  (Thermo Fisher)


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    Name:
    IgM Antibody
    Description:
    Designed for immunofluorescence staining Thermo Scientific IgM FITC Labeled Antibody allows rapid and inexpensive assessment of tissues
    Catalog Number:
    RB-1922-R2
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
    Applications:
    Anatomical Pathology|Clinical
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    Structured Review

    Thermo Fisher igm
    Resveratrol inhibited T cell activation and antigen-specific antibody production in vivo. ( a ) Collagen-specific <t>IgM</t> in the serum from immunized mice was detected by ELISA. ( b ) The proliferation of collagen-specific T cell prepared from collagen-immunized mice spleen were measured through BrdU proliferation assay. ( c – f ) The <t>cytokines</t> in the serum from mice immunized with collagen were detected by ELISA (* P
    Designed for immunofluorescence staining Thermo Scientific IgM FITC Labeled Antibody allows rapid and inexpensive assessment of tissues
    https://www.bioz.com/result/igm/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
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    igm - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "Resveratrol Inhibits CD4+ T Cell Activation by Enhancing the Expression and Activity of Sirt1"

    Article Title: Resveratrol Inhibits CD4+ T Cell Activation by Enhancing the Expression and Activity of Sirt1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075139

    Resveratrol inhibited T cell activation and antigen-specific antibody production in vivo. ( a ) Collagen-specific IgM in the serum from immunized mice was detected by ELISA. ( b ) The proliferation of collagen-specific T cell prepared from collagen-immunized mice spleen were measured through BrdU proliferation assay. ( c – f ) The cytokines in the serum from mice immunized with collagen were detected by ELISA (* P
    Figure Legend Snippet: Resveratrol inhibited T cell activation and antigen-specific antibody production in vivo. ( a ) Collagen-specific IgM in the serum from immunized mice was detected by ELISA. ( b ) The proliferation of collagen-specific T cell prepared from collagen-immunized mice spleen were measured through BrdU proliferation assay. ( c – f ) The cytokines in the serum from mice immunized with collagen were detected by ELISA (* P

    Techniques Used: Activation Assay, In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Proliferation Assay

    2) Product Images from "Neonatal Exposure to Pneumococcal Phosphorylcholine Modulates the Development of House Dust Mite Allergy during Adult Life"

    Article Title: Neonatal Exposure to Pneumococcal Phosphorylcholine Modulates the Development of House Dust Mite Allergy during Adult Life

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1500251

    IgM-expressing B cells dominated the local immune response to HDM among mice immunized with PC-bearing R36A as neonates. Following exposure to HDM, lungs were perfused and enzymatically digested to enumerate B cells in the lung. ( A ) B220 + CD19 + cells that ( B and C ) expressed IgM as well as ( D ) CD138 + B220 low cells that expressed IgM were quantified and also identified from the lung by ( G . Cells isolated from perfused lungs were incubated on anti-IgM–coated plates for ELISPOT analysis and were ( E and F ) visually enumerated. ( I – L ) Cryosections of lungs from these mice were stained for IgM (green) and laminin (gray), and bronchioles (Br) and vessels (v) are labeled as such. These ( H ) IgM + cells were quantified from 50 fields per section. Values represent the mean ± SEM from three to five independent experiments with 5–10 mice per group. Data were analyzed by ANOVA, in which statistically significant results are represented as * p
    Figure Legend Snippet: IgM-expressing B cells dominated the local immune response to HDM among mice immunized with PC-bearing R36A as neonates. Following exposure to HDM, lungs were perfused and enzymatically digested to enumerate B cells in the lung. ( A ) B220 + CD19 + cells that ( B and C ) expressed IgM as well as ( D ) CD138 + B220 low cells that expressed IgM were quantified and also identified from the lung by ( G . Cells isolated from perfused lungs were incubated on anti-IgM–coated plates for ELISPOT analysis and were ( E and F ) visually enumerated. ( I – L ) Cryosections of lungs from these mice were stained for IgM (green) and laminin (gray), and bronchioles (Br) and vessels (v) are labeled as such. These ( H ) IgM + cells were quantified from 50 fields per section. Values represent the mean ± SEM from three to five independent experiments with 5–10 mice per group. Data were analyzed by ANOVA, in which statistically significant results are represented as * p

    Techniques Used: Expressing, Mouse Assay, Isolation, Incubation, Enzyme-linked Immunospot, Staining, Labeling

    Neonatal exposure to PC-bearing R36A resulted in the accumulation of CD138 + IgM-expressing PC-specific cells in the lung following exposure to HDM. Following exposure to HDM, lungs were perfused and enzymatically digested. Cells isolated from perfused lungs were incubated on PC-coated plates for ELISPOT analysis and were ( A and B ) visually enumerated. ( C ) The first milliliter of the BALF was collected to quantify (A) anti-PC IgM by ELISA. ( D and E . These PC-specific B cells were also stained for ( F ) CD138. ( G – J ) Cryosections of lungs from these groups of mice were also stained for IgM (green), CD138 (red), PC-BSA binding (yellow), and laminin (gray) and viewed with a Leica/Leitz DMRB microscope. Values represent the mean ± SEM from three to five independent experiments with 5–10 mice per group. Data were analyzed by ANOVA, in which statistically significant results are represented as * p
    Figure Legend Snippet: Neonatal exposure to PC-bearing R36A resulted in the accumulation of CD138 + IgM-expressing PC-specific cells in the lung following exposure to HDM. Following exposure to HDM, lungs were perfused and enzymatically digested. Cells isolated from perfused lungs were incubated on PC-coated plates for ELISPOT analysis and were ( A and B ) visually enumerated. ( C ) The first milliliter of the BALF was collected to quantify (A) anti-PC IgM by ELISA. ( D and E . These PC-specific B cells were also stained for ( F ) CD138. ( G – J ) Cryosections of lungs from these groups of mice were also stained for IgM (green), CD138 (red), PC-BSA binding (yellow), and laminin (gray) and viewed with a Leica/Leitz DMRB microscope. Values represent the mean ± SEM from three to five independent experiments with 5–10 mice per group. Data were analyzed by ANOVA, in which statistically significant results are represented as * p

    Techniques Used: Expressing, Isolation, Incubation, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Staining, Mouse Assay, Binding Assay, Microscopy

    3) Product Images from "Rituximab Treatment Prevents the Early Development of Proteinuria following Pig-to-Baboon Xeno-Kidney Transplantation"

    Article Title: Rituximab Treatment Prevents the Early Development of Proteinuria following Pig-to-Baboon Xeno-Kidney Transplantation

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2013040363

    Onset of post-transplant proteinuria, expression of SMPDL-3b in the xeno kidney grafts, and levels of anti-pig IgG and IgM. (A) Onset of 2+ proteinuria in animals treated with rituximab after xeno-kidney transplantation (KTx; red dots). (B) SMPDL-3b expression
    Figure Legend Snippet: Onset of post-transplant proteinuria, expression of SMPDL-3b in the xeno kidney grafts, and levels of anti-pig IgG and IgM. (A) Onset of 2+ proteinuria in animals treated with rituximab after xeno-kidney transplantation (KTx; red dots). (B) SMPDL-3b expression

    Techniques Used: Expressing, Transplantation Assay

    4) Product Images from "Spontaneous Production of Immunoglobulin M in Human Epithelial Cancer Cells"

    Article Title: Spontaneous Production of Immunoglobulin M in Human Epithelial Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051423

    TLR9 agonists stimulated human epithelial cancer cells to secrete IgM. A, Ig µ mRNA level was analyzed by semiquantitative RT-PCR after stimulation by CpG 2006, and the two non-CpG ODN controls, CpG 2078 and GpC. CpG-N ODN208, as negative control; GAPDH, internal control. B, flow cytometry analysis showed that cytoplasmic IgM was decreased after stimulation with CpG 2006, CpG 2078, and GpC. CpG-N ODN208, as negative control. ** P
    Figure Legend Snippet: TLR9 agonists stimulated human epithelial cancer cells to secrete IgM. A, Ig µ mRNA level was analyzed by semiquantitative RT-PCR after stimulation by CpG 2006, and the two non-CpG ODN controls, CpG 2078 and GpC. CpG-N ODN208, as negative control; GAPDH, internal control. B, flow cytometry analysis showed that cytoplasmic IgM was decreased after stimulation with CpG 2006, CpG 2078, and GpC. CpG-N ODN208, as negative control. ** P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Gel Permeation Chromatography, Negative Control, Flow Cytometry, Cytometry

    Chloroquine abolished effects for TLR9 agonists on IgM secretion in HeLa MR cells. A, semi-quantative RT-PCR showed increased Ig µ expression after stimulation by CpG 2006, CpG 2078 and GpC (upper panel). The effects of TLR9 agonists were abolished by chloroquine (lower panel). GAPDH, internal control. B, flow cytometry analysis showed that the cytoplasmic IgM level was decreased after stimulation with CpG 2006, CpG 2078 and GpC, and that this was abolished by chloroquine. * P
    Figure Legend Snippet: Chloroquine abolished effects for TLR9 agonists on IgM secretion in HeLa MR cells. A, semi-quantative RT-PCR showed increased Ig µ expression after stimulation by CpG 2006, CpG 2078 and GpC (upper panel). The effects of TLR9 agonists were abolished by chloroquine (lower panel). GAPDH, internal control. B, flow cytometry analysis showed that the cytoplasmic IgM level was decreased after stimulation with CpG 2006, CpG 2078 and GpC, and that this was abolished by chloroquine. * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Gel Permeation Chromatography, Flow Cytometry, Cytometry

    Knockdown of MyD88 by siRNA inhibited TLR9 agonist-induced IgM secretion in HeLa MR cells. A, effects of three synthesized siRNAs for knocking down MyD88 expression were analyzed after transfection from 24 h to 96 h by semiquantitative RT-PCR. Results of the effective siRNAs 1 and 3 are shown. B, semi-quantative RT-PCR showed increased Ig µ expression after stimulation by CpG 2006, CpG 2078 and GpC (upper panel). This was abolished by MyD88 knockdown with siRNA1 and siRNA3 (lower panel). GAPDH, internal control. C, flow cytometry analysis showed that the cytoplasmic IgM level was decreased after stimulation with CpG 2006, CpG 2078 and GpC, and that this was abolished by MyD88 knockdown with siRNA1 and siRNA3. ** P
    Figure Legend Snippet: Knockdown of MyD88 by siRNA inhibited TLR9 agonist-induced IgM secretion in HeLa MR cells. A, effects of three synthesized siRNAs for knocking down MyD88 expression were analyzed after transfection from 24 h to 96 h by semiquantitative RT-PCR. Results of the effective siRNAs 1 and 3 are shown. B, semi-quantative RT-PCR showed increased Ig µ expression after stimulation by CpG 2006, CpG 2078 and GpC (upper panel). This was abolished by MyD88 knockdown with siRNA1 and siRNA3 (lower panel). GAPDH, internal control. C, flow cytometry analysis showed that the cytoplasmic IgM level was decreased after stimulation with CpG 2006, CpG 2078 and GpC, and that this was abolished by MyD88 knockdown with siRNA1 and siRNA3. ** P

    Techniques Used: Synthesized, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Gel Permeation Chromatography, Flow Cytometry, Cytometry

    5) Product Images from "Fate mapping quantifies the dynamics of B cell development and activation throughout life"

    Article Title: Fate mapping quantifies the dynamics of B cell development and activation throughout life

    Journal: bioRxiv

    doi: 10.1101/871624

    Variation in the degree of stable chimerism in B cell subsets within the same mice. (A) Variation in the chimerism among AA4.1 + IgM hi IgD − B cell progenitors and recirculating FM B cells within different BM sites. (B) Variation in chimerism across different lymph nodes. Data from two representative animals.
    Figure Legend Snippet: Variation in the degree of stable chimerism in B cell subsets within the same mice. (A) Variation in the chimerism among AA4.1 + IgM hi IgD − B cell progenitors and recirculating FM B cells within different BM sites. (B) Variation in chimerism across different lymph nodes. Data from two representative animals.

    Techniques Used: Mouse Assay

    6) Product Images from "A Robust and Versatile Automated Glycoanalytical Technology for Serum Antibodies and Acute Phase Proteins: Ovarian Cancer Case Study *"

    Article Title: A Robust and Versatile Automated Glycoanalytical Technology for Serum Antibodies and Acute Phase Proteins: Ovarian Cancer Case Study *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA119.001531

    Glycosylation traits for selected antibodies (IgG, IgM, IgA) and acute phase proteins (Trf, Hpt, A1AT) for healthy human serum are presented (5 A ). . Glycosylation features of antibodies (IgG, IgM and IgA) and acute phase proteins (Trf, Hpt, A1AT) are presented as control charts (5 B and 5 C ).
    Figure Legend Snippet: Glycosylation traits for selected antibodies (IgG, IgM, IgA) and acute phase proteins (Trf, Hpt, A1AT) for healthy human serum are presented (5 A ). . Glycosylation features of antibodies (IgG, IgM and IgA) and acute phase proteins (Trf, Hpt, A1AT) are presented as control charts (5 B and 5 C ).

    Techniques Used:

    UPLC-HILIC-FLD chromatograms of AQC labeled N -glycans released from human serum for affinity purified glycoproteins IgG, IgM and IgA, Trf, Hpt, A1AT and 2-AB labeled N -glycans released from total serum. Only major glycans are annotated. All major N -glycans identified within the total serum UPLC chromatogram are present in the individual glycoprotein chromatograms which showcases that serum glycosylation is largely dominated by the highest abundance proteins (IgG, IgM and IgA, Trf, Hpt and A1AT). SNFG glycan nomenclature is used throughout.
    Figure Legend Snippet: UPLC-HILIC-FLD chromatograms of AQC labeled N -glycans released from human serum for affinity purified glycoproteins IgG, IgM and IgA, Trf, Hpt, A1AT and 2-AB labeled N -glycans released from total serum. Only major glycans are annotated. All major N -glycans identified within the total serum UPLC chromatogram are present in the individual glycoprotein chromatograms which showcases that serum glycosylation is largely dominated by the highest abundance proteins (IgG, IgM and IgA, Trf, Hpt and A1AT). SNFG glycan nomenclature is used throughout.

    Techniques Used: Hydrophilic Interaction Liquid Chromatography, Labeling, Affinity Purification

    Multiplexed automated serial capture of glycoprotein N -glycoprofiling. 96-well format robotic platform ( A ), specific anti-glycoprotein capture resin packed in PhyNexus phytip ( B ), serial capture of selected glycoproteins 1.Trf, 2.IgG, 3.IgM, 4.IgA, 5.Hpt, 6.A1AT ( C ), 1D SDS-PAGE separation of multiplexed automated capture of six selected glycoproteins from pooled human serum using PhyNexus Phytips. Lane 1: Protein Marker, Lane 2: Trf Standard, Lane 3: Bound Trf, Lane 4: Bound IgG, Lane 5: Bound IgM, Lane 6: Bound IgA, Lane 7: A1AT Standard, Lane 8: Bound A1AT, Lane 9: Hpt Standard, Lane 10: Bound Hpt. The protein bands marked with arrows are traces of albumin protein (non-glycosylated) ( D ), automated glycoprotein sample preparation, PNGaseF release and aminoquinoline carbamate (AQC) labeling of N -glycans ( E ) and finally ultra-high performance liquid chromatography (UPLC) separation and glycan structural analysis ( F ).
    Figure Legend Snippet: Multiplexed automated serial capture of glycoprotein N -glycoprofiling. 96-well format robotic platform ( A ), specific anti-glycoprotein capture resin packed in PhyNexus phytip ( B ), serial capture of selected glycoproteins 1.Trf, 2.IgG, 3.IgM, 4.IgA, 5.Hpt, 6.A1AT ( C ), 1D SDS-PAGE separation of multiplexed automated capture of six selected glycoproteins from pooled human serum using PhyNexus Phytips. Lane 1: Protein Marker, Lane 2: Trf Standard, Lane 3: Bound Trf, Lane 4: Bound IgG, Lane 5: Bound IgM, Lane 6: Bound IgA, Lane 7: A1AT Standard, Lane 8: Bound A1AT, Lane 9: Hpt Standard, Lane 10: Bound Hpt. The protein bands marked with arrows are traces of albumin protein (non-glycosylated) ( D ), automated glycoprotein sample preparation, PNGaseF release and aminoquinoline carbamate (AQC) labeling of N -glycans ( E ) and finally ultra-high performance liquid chromatography (UPLC) separation and glycan structural analysis ( F ).

    Techniques Used: SDS Page, Marker, Sample Prep, Labeling, High Performance Liquid Chromatography

    Serial capture and N -glycoprofiling of human serum IgG, IgM and IgA, purified from human serum. The 25 IgG glycan peak areas (G1-G25), 24 IgM peak areas (M1–24) and 25 IgA glycan peak areas (A1-A25) plotted for five technical replicates over three different days (3 A ). The standard error shown as error bars. Sequencing of AQC labeled IgG, IgM and IgA N -glycans visualized by UPLC-HILIC chromatograms using exoglycosidase enzymes with glucose units (GU) to facilitate glycan identification (3 B ). Digestion of AQC labeled IgG, IgM and IgA N -glycans with addition of sialidase (ABS), galactosidase (BTG), hexosaminidase (GUH), fucosidase (BKF) and mannosidase (JBM) in the following order ABS, ABS+BTG, ABS+BTG+GUH, ABS+BTG+GUH+BKF and ABS+BTG+GUH+BKF+JBM. For IgG, arrows indicate the cleavage of sugar residues for selected peaks: major glycans FA2G2S1 and FA2G2S2. For IgM, arrows indicate the cleavage of sugar residues for selected peaks: major glycans FA2G2S1 and FA2BG2S1. For IgA, arrows indicate the cleavage of sugar residues for selected peaks: major glycans A2G2S1, FA2G2S2 and FA2BG2S2. SNFG nomenclature is used for glycan representation.
    Figure Legend Snippet: Serial capture and N -glycoprofiling of human serum IgG, IgM and IgA, purified from human serum. The 25 IgG glycan peak areas (G1-G25), 24 IgM peak areas (M1–24) and 25 IgA glycan peak areas (A1-A25) plotted for five technical replicates over three different days (3 A ). The standard error shown as error bars. Sequencing of AQC labeled IgG, IgM and IgA N -glycans visualized by UPLC-HILIC chromatograms using exoglycosidase enzymes with glucose units (GU) to facilitate glycan identification (3 B ). Digestion of AQC labeled IgG, IgM and IgA N -glycans with addition of sialidase (ABS), galactosidase (BTG), hexosaminidase (GUH), fucosidase (BKF) and mannosidase (JBM) in the following order ABS, ABS+BTG, ABS+BTG+GUH, ABS+BTG+GUH+BKF and ABS+BTG+GUH+BKF+JBM. For IgG, arrows indicate the cleavage of sugar residues for selected peaks: major glycans FA2G2S1 and FA2G2S2. For IgM, arrows indicate the cleavage of sugar residues for selected peaks: major glycans FA2G2S1 and FA2BG2S1. For IgA, arrows indicate the cleavage of sugar residues for selected peaks: major glycans A2G2S1, FA2G2S2 and FA2BG2S2. SNFG nomenclature is used for glycan representation.

    Techniques Used: Purification, Sequencing, Labeling, Hydrophilic Interaction Liquid Chromatography

    7) Product Images from "Altered BCR signalling quality predisposes to autoimmune disease and a pre-diabetic state"

    Article Title: Altered BCR signalling quality predisposes to autoimmune disease and a pre-diabetic state

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2012.169

    Reduced Syk-family kinase activity in Syk ki cells translates into attenuated anti-IgM-induced phosphotyrosine signalling and a diminished B lymphocytic Ca 2+ response. ( A , left) To compare their auto- and transphosphorylation capacity, HA:Syk and HA:Zap-70 were stably expressed in CHO cells at comparable, low levels as shown by anti-HA immunoprecipitation (IP) and western blotting (WB). A kinase-dead mutant (KD) of Syk, HA:Syk(K402R) served as negative control. Associated kinase activity was assayed by incubation of a part of the anti-HA immunoprecipitated material in vitro in the presence of Mg 2+ , γ-ATP- 32 P and recombinant GST-Igβ ( A , right) for the indicated time points. ( B ) To assess the phosphorylation state of Igβ in vivo , splenic B cells from Syk wt and Syk ki mice were left untreated (0) or stimulated with anti-IgM (anti-μ) for 2 min; subsequently, Igβ (red arrows) was immunoprecipitated and the amount of total Igβ and p-Igβ (4G10) was determined. ( C ) Phosphotyrosine levels (4G10) of total ctrl (B220 + CD19 + ), T2/MZ ctrl (B220 + CD21 hi CD24 hi ) and total ki (B220 + CD19 + ) B cell lysates after anti-IgM F(ab′)2-mediated stimulation. ( D ) Reduced anti-IgM-induced calcium responses of fluo-4 AM-loaded splenic B220 + CD19 + ki B cells; black arrows indicate the addition of anti-IgM at concentrations depicted above response curves; red arrows mark the addition of extracellular Ca 2+ .
    Figure Legend Snippet: Reduced Syk-family kinase activity in Syk ki cells translates into attenuated anti-IgM-induced phosphotyrosine signalling and a diminished B lymphocytic Ca 2+ response. ( A , left) To compare their auto- and transphosphorylation capacity, HA:Syk and HA:Zap-70 were stably expressed in CHO cells at comparable, low levels as shown by anti-HA immunoprecipitation (IP) and western blotting (WB). A kinase-dead mutant (KD) of Syk, HA:Syk(K402R) served as negative control. Associated kinase activity was assayed by incubation of a part of the anti-HA immunoprecipitated material in vitro in the presence of Mg 2+ , γ-ATP- 32 P and recombinant GST-Igβ ( A , right) for the indicated time points. ( B ) To assess the phosphorylation state of Igβ in vivo , splenic B cells from Syk wt and Syk ki mice were left untreated (0) or stimulated with anti-IgM (anti-μ) for 2 min; subsequently, Igβ (red arrows) was immunoprecipitated and the amount of total Igβ and p-Igβ (4G10) was determined. ( C ) Phosphotyrosine levels (4G10) of total ctrl (B220 + CD19 + ), T2/MZ ctrl (B220 + CD21 hi CD24 hi ) and total ki (B220 + CD19 + ) B cell lysates after anti-IgM F(ab′)2-mediated stimulation. ( D ) Reduced anti-IgM-induced calcium responses of fluo-4 AM-loaded splenic B220 + CD19 + ki B cells; black arrows indicate the addition of anti-IgM at concentrations depicted above response curves; red arrows mark the addition of extracellular Ca 2+ .

    Techniques Used: Activity Assay, Stable Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Negative Control, Incubation, In Vitro, Recombinant, In Vivo, Mouse Assay

    Age-related accumulation of Syk ki marginal zone B cells. ( A ) B220 + pre-gated splenic cells were stained with anti-IgM and anti-IgD to delineate transitional and mature B cell fractions. ( B ) Statistical analysis of total B220 + , transitional (T) 1/marginal zone (MZ), T2 and mature (M) splenic B cell and ( C ) CD4 + and CD8 + (CD3 + -pre-gated) T cell numbers in ctrl and ki mice. ( D ) Numbers of (CD3 + -pre-gated) CD44 hi CD62L lo effector/memory CD4 + and CD8 + T cell subsets and ( E ) ratio of splenic CD4 + effector/memory T versus CD4 + CD25 + FoxP3 + nTreg cells. ( F , left) Representative dot plot of MZ (B220 + CD21 hi CD23 lo/ − ) and follicular (FO)/T2 (B220 + CD21 int CD23 + ) B cells of ctrl mice at the age of 8 weeks (W); numbers in dot plots represent average percentages of B cell subsets. ( F , right, G ) Age-related accumulation of B220 + CD21 hi CD23 lo/ − MZ B cells in ki mice. ( H ) Pre-immune serum immunoglobulin (Ig) titres of ctrl and ki mice. For ( A – E , H ), mice were analysed at the age of 8–12 weeks. * P
    Figure Legend Snippet: Age-related accumulation of Syk ki marginal zone B cells. ( A ) B220 + pre-gated splenic cells were stained with anti-IgM and anti-IgD to delineate transitional and mature B cell fractions. ( B ) Statistical analysis of total B220 + , transitional (T) 1/marginal zone (MZ), T2 and mature (M) splenic B cell and ( C ) CD4 + and CD8 + (CD3 + -pre-gated) T cell numbers in ctrl and ki mice. ( D ) Numbers of (CD3 + -pre-gated) CD44 hi CD62L lo effector/memory CD4 + and CD8 + T cell subsets and ( E ) ratio of splenic CD4 + effector/memory T versus CD4 + CD25 + FoxP3 + nTreg cells. ( F , left) Representative dot plot of MZ (B220 + CD21 hi CD23 lo/ − ) and follicular (FO)/T2 (B220 + CD21 int CD23 + ) B cells of ctrl mice at the age of 8 weeks (W); numbers in dot plots represent average percentages of B cell subsets. ( F , right, G ) Age-related accumulation of B220 + CD21 hi CD23 lo/ − MZ B cells in ki mice. ( H ) Pre-immune serum immunoglobulin (Ig) titres of ctrl and ki mice. For ( A – E , H ), mice were analysed at the age of 8–12 weeks. * P

    Techniques Used: Staining, Mouse Assay

    Syk ki mice produce anti-insulin autoantibodies and develop age-related glomerulonephritis. ( A ) Permeabilized Hep-2 cells were incubated with Syk ctrl or Syk ki mouse sera, stained with anti-IgM-FITC or anti-IgG-FITC and analysed for Hep-2 reactivity via epifluorescence microscopy (Zeiss Axioscope, × 10, scale bar=30 μm). ( A , left image and middle panel) Representative images indicating prominent cytoplasmic Hep-2 reactivity (IgM depicted) of ki sera. ( A , right) Numbers below pie charts indicate Hep-2 reactive sera (IgM + and IgG + )/total number of animals analysed. ( B ) Kidney cryosections of ctrl and ki mice (8 weeks) were stained with anti-IgM-FITC (upper panel), anti-IgG-FITC (lower panel) and anti-C3 to assess glomerular immune complex and complement deposition (× 20/ × 63, scale bar=100 μm). ( C ) Periodic acid Schiff staining of kidney paraffin sections from Syk ctrl (i, ii) and Syk ki (iii, iv) mice at the age of 60 weeks (× 63, scale bar=50 μm) showed significant Syk ki glomerular mesangial hypercellularity (*), mesangial matrix accumulation (arrows) and basement membrane increase (**). ( D ) Quantification of glomerular diameter and ( E ) proteinuria in Syk ctrl and Syk ki mice. ( F ) dsDNA-specific IgM titres were comparable between Syk ctrl and Syk ki mice, while ( G ) insulin-specific IgM titres were elevated in ki mice (8–12 weeks). ( H ) Basal blood glucose levels of 14-week-old Syk ctrl and Syk ki mice were measured after 16 h of chow starvation and ( I ) 0, 15, 30, 60 and 120 min after intraperitoneal injection of D-glucose (2 g/kg). ( J ) To test whether the anti-insulin-specific IgM titres in Syk ki mice were aggravated upon TLR-4 stimulation of MZ B cells, sorted B220 + CD19 + splenic B cells were incubated with LPS for 36 h and analysed for spot enlargement upon stimulation. * P
    Figure Legend Snippet: Syk ki mice produce anti-insulin autoantibodies and develop age-related glomerulonephritis. ( A ) Permeabilized Hep-2 cells were incubated with Syk ctrl or Syk ki mouse sera, stained with anti-IgM-FITC or anti-IgG-FITC and analysed for Hep-2 reactivity via epifluorescence microscopy (Zeiss Axioscope, × 10, scale bar=30 μm). ( A , left image and middle panel) Representative images indicating prominent cytoplasmic Hep-2 reactivity (IgM depicted) of ki sera. ( A , right) Numbers below pie charts indicate Hep-2 reactive sera (IgM + and IgG + )/total number of animals analysed. ( B ) Kidney cryosections of ctrl and ki mice (8 weeks) were stained with anti-IgM-FITC (upper panel), anti-IgG-FITC (lower panel) and anti-C3 to assess glomerular immune complex and complement deposition (× 20/ × 63, scale bar=100 μm). ( C ) Periodic acid Schiff staining of kidney paraffin sections from Syk ctrl (i, ii) and Syk ki (iii, iv) mice at the age of 60 weeks (× 63, scale bar=50 μm) showed significant Syk ki glomerular mesangial hypercellularity (*), mesangial matrix accumulation (arrows) and basement membrane increase (**). ( D ) Quantification of glomerular diameter and ( E ) proteinuria in Syk ctrl and Syk ki mice. ( F ) dsDNA-specific IgM titres were comparable between Syk ctrl and Syk ki mice, while ( G ) insulin-specific IgM titres were elevated in ki mice (8–12 weeks). ( H ) Basal blood glucose levels of 14-week-old Syk ctrl and Syk ki mice were measured after 16 h of chow starvation and ( I ) 0, 15, 30, 60 and 120 min after intraperitoneal injection of D-glucose (2 g/kg). ( J ) To test whether the anti-insulin-specific IgM titres in Syk ki mice were aggravated upon TLR-4 stimulation of MZ B cells, sorted B220 + CD19 + splenic B cells were incubated with LPS for 36 h and analysed for spot enlargement upon stimulation. * P

    Techniques Used: Mouse Assay, Incubation, Staining, Epifluorescence Microscopy, Injection

    8) Product Images from "Binding of Plasmodium falciparum Merozoite Surface Proteins DBLMSP and DBLMSP2 to Human Immunoglobulin M Is Conserved among Broadly Diverged Sequence Variants *"

    Article Title: Binding of Plasmodium falciparum Merozoite Surface Proteins DBLMSP and DBLMSP2 to Human Immunoglobulin M Is Conserved among Broadly Diverged Sequence Variants *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.722074

    Human IgM bind native DBLMSP protein. A , indirect immunofluorescence assay for detection of human IgM was conducted on live wild-type and Δ dblmsp merozoites grown in either 20% human serum (+ HS ) or 0.125 mg/ml purified human IgM (+ IgM ). Alexa Fluor 488 goat anti-human IgM μ chain antibodies are depicted in green in panel 1. Panel 2 corresponds to the bright field image, and panel 3 shows the overlay. B , immunoelectron microscopy showing the presence of human IgM at the surface of wild-type but not Δ dblmsp purified merozoites. Scale bar represents 200 nm. C , addition of 1 mg/ml purified human IgM to both wild-type and Δ dblmsp parasites grown in Albumax did not affect their ability to infect red blood cells. Bars represent means ± S.D.; n = 3 replicate wells. A representative of four independent experiments is presented.
    Figure Legend Snippet: Human IgM bind native DBLMSP protein. A , indirect immunofluorescence assay for detection of human IgM was conducted on live wild-type and Δ dblmsp merozoites grown in either 20% human serum (+ HS ) or 0.125 mg/ml purified human IgM (+ IgM ). Alexa Fluor 488 goat anti-human IgM μ chain antibodies are depicted in green in panel 1. Panel 2 corresponds to the bright field image, and panel 3 shows the overlay. B , immunoelectron microscopy showing the presence of human IgM at the surface of wild-type but not Δ dblmsp purified merozoites. Scale bar represents 200 nm. C , addition of 1 mg/ml purified human IgM to both wild-type and Δ dblmsp parasites grown in Albumax did not affect their ability to infect red blood cells. Bars represent means ± S.D.; n = 3 replicate wells. A representative of four independent experiments is presented.

    Techniques Used: Immunofluorescence, Purification, Immuno-Electron Microscopy

    9) Product Images from "Antibody production in autoimmune BXSB mice. I. CD40L-expressing B cells need fewer signals for polyclonal antibody synthesis"

    Article Title: Antibody production in autoimmune BXSB mice. I. CD40L-expressing B cells need fewer signals for polyclonal antibody synthesis

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.1999.01032.x

    CD40-Ig blocks total immunoglobulin and anti-ssDNA production by large B cells from male BXSB mice. (a) Large B cells from male BXSB mice were isolated and cultured with IL-4/IL-5 in the presence or absence of 0.3 ng/ml anti-IgD-dextran. CD40-Ig (□, 25 μg/ml) or control immunoglobulin (▪) was added at the initiation of culture. Culture supernatants were collected at 120 h and analysed for the presence of IgM and IgG1 by ELISA. (b) Large B cells from male BXSB mice were isolated and cultured with IL-4/IL-5 in the presence of 0.3 ng/ml anti-IgD-dextran. CD40-Ig (□, 25 μg/ml) or control immunoglobulin (▪) was added at the initiation of culture. Culture supernatants were collected at 120 h and analysed for the presence of anti-ssDNA antibody by ELISA. *Significantly different ( P
    Figure Legend Snippet: CD40-Ig blocks total immunoglobulin and anti-ssDNA production by large B cells from male BXSB mice. (a) Large B cells from male BXSB mice were isolated and cultured with IL-4/IL-5 in the presence or absence of 0.3 ng/ml anti-IgD-dextran. CD40-Ig (□, 25 μg/ml) or control immunoglobulin (▪) was added at the initiation of culture. Culture supernatants were collected at 120 h and analysed for the presence of IgM and IgG1 by ELISA. (b) Large B cells from male BXSB mice were isolated and cultured with IL-4/IL-5 in the presence of 0.3 ng/ml anti-IgD-dextran. CD40-Ig (□, 25 μg/ml) or control immunoglobulin (▪) was added at the initiation of culture. Culture supernatants were collected at 120 h and analysed for the presence of anti-ssDNA antibody by ELISA. *Significantly different ( P

    Techniques Used: Mouse Assay, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

    Small and large B cells from male BXSB mice have fewer requirements for total antibody production than small and large B cells from female BXSB mice. Small and large B cells from male (▪) and female (□) BXSB mice were cultured either with IL-4 and IL-5 alone, or in the presence of increasing concentrations of anti-IgD-dextran ± anti-CD40. Culture supernatants (six wells/group) were collected at 120 h and analysed for the presence of IgM and IgG1 by ELISA. The values presented are the means ± s.d. from three experiments. *Significantly different ( P
    Figure Legend Snippet: Small and large B cells from male BXSB mice have fewer requirements for total antibody production than small and large B cells from female BXSB mice. Small and large B cells from male (▪) and female (□) BXSB mice were cultured either with IL-4 and IL-5 alone, or in the presence of increasing concentrations of anti-IgD-dextran ± anti-CD40. Culture supernatants (six wells/group) were collected at 120 h and analysed for the presence of IgM and IgG1 by ELISA. The values presented are the means ± s.d. from three experiments. *Significantly different ( P

    Techniques Used: Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    10) Product Images from "Hypomorphic Mutations in the BCR Signalosome Lead to Selective Immunoglobulin M Deficiency and Impaired B-cell Homeostasis"

    Article Title: Hypomorphic Mutations in the BCR Signalosome Lead to Selective Immunoglobulin M Deficiency and Impaired B-cell Homeostasis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02984

    Molecular characterization of novel hypomorphic BTK/BLNK mutations. (A) Gene expression of precursor, secreted and membrane IgM of patients and control EBV-LCL quantified by semi-quantitative cDNA-PCR. (B) Protein expression of monomeric and pentameric IgM of patients and control EBV-LCLs by SDS-PAGE or native-PAGE and detected by western blot. (C) BTK/BLNK Mutation analysis of genomic DNA from peripheral blood. Patient A is hemizygote for a c615G > T in BTK. Patient B is compound heterozygous in BLNK for c328C > G, c472G > T. Healthy controls served as wild type control. (D) Schematic depiction of mutation sites and phosphorylation sites in BTK and BLNK.
    Figure Legend Snippet: Molecular characterization of novel hypomorphic BTK/BLNK mutations. (A) Gene expression of precursor, secreted and membrane IgM of patients and control EBV-LCL quantified by semi-quantitative cDNA-PCR. (B) Protein expression of monomeric and pentameric IgM of patients and control EBV-LCLs by SDS-PAGE or native-PAGE and detected by western blot. (C) BTK/BLNK Mutation analysis of genomic DNA from peripheral blood. Patient A is hemizygote for a c615G > T in BTK. Patient B is compound heterozygous in BLNK for c328C > G, c472G > T. Healthy controls served as wild type control. (D) Schematic depiction of mutation sites and phosphorylation sites in BTK and BLNK.

    Techniques Used: Expressing, Polymerase Chain Reaction, SDS Page, Clear Native PAGE, Western Blot, Mutagenesis

    11) Product Images from "Th2-like T-follicular helper cells promote functional antibody production during Plasmodium falciparum infection"

    Article Title: Th2-like T-follicular helper cells promote functional antibody production during Plasmodium falciparum infection

    Journal: bioRxiv

    doi: 10.1101/2020.05.18.101048

    Antibodies to merozoite and MSP2 were induced following IBSM A-D) IgG subclasses and IgM responses and functional capacity to fix complement (C1q), binding of dimeric Fc γ RIIa or Fc γ RIIIa (surrogates for IgG antibody capacity to cross-link cellular receptors) and to promote opsonic phagocytosis (OPA) to merozoites (A/C) and MSP2 (B/D) were assessed prior to inoculation and at end-of-study time points (EOS). Positive threshold for each responses is indicated by dotted line (calculated as mean + 3 SD of day 0 responses and the proportion of positive responders is indicated in the bottom right of each panel. Wilcoxon rank-sign t test is
    Figure Legend Snippet: Antibodies to merozoite and MSP2 were induced following IBSM A-D) IgG subclasses and IgM responses and functional capacity to fix complement (C1q), binding of dimeric Fc γ RIIa or Fc γ RIIIa (surrogates for IgG antibody capacity to cross-link cellular receptors) and to promote opsonic phagocytosis (OPA) to merozoites (A/C) and MSP2 (B/D) were assessed prior to inoculation and at end-of-study time points (EOS). Positive threshold for each responses is indicated by dotted line (calculated as mean + 3 SD of day 0 responses and the proportion of positive responders is indicated in the bottom right of each panel. Wilcoxon rank-sign t test is

    Techniques Used: Functional Assay, Binding Assay

    12) Product Images from "Sex-specific differences in the function and differentiation of ABCs mark TLR7-driven immunopathogenesis"

    Article Title: Sex-specific differences in the function and differentiation of ABCs mark TLR7-driven immunopathogenesis

    Journal: bioRxiv

    doi: 10.1101/2021.01.20.427400

    IRF8 and IRF5 are required for ABC generation and differentiation in DKO females. (A-C) Quantifications showing the numbers of CD11c + Tbet + ABCs (A) , CD11c + CD11b + ABCs (B) , and total GL7 + Fas + B cells (C) from the spleens of WT, DKO(F), CD23- Irf8 .DKO(F), CD23- Irf5 f/+ .DKO(F), and CD23- Irf5. DKO(F) mice. Data pooled from at least 5 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (D) Representative immunofluorescence images of B220 (blue), GL7 (pink) , and CD3 (green) on the indicated mice. Data representative of at least 4 mice per genotype and show mean +/− SEM; p-value by unpaired two-tailed t-test. (E-F) Quantifications showing the numbers of total PB/PCs (B220 mid/lo CD138 + ) (E) and CD11c + PB/PCs (F) from the indicated mice. Data pooled from at least 5 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (G) ELISA data showing anti-dsDNA IgG2c in the serum from the indicated mice. Data pooled from at least 4 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (H-K) CD23 + B cells from the indicated mice were stimulated for 2-3d with α IgM, α CD40, and IL-21. (H) Quantification of CD11c + Tbet + B cells after 3d culture. Data pooled from at least 3 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (I) Quantifications showing the expression of CD11c and Tbet in the indicated strains after 3d culture. Data pooled from at least 4 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (J) ELISA data showing CXCL10 production in the supernatants after 3d culture. Data representative of 3 independent experiments and show mean +/− SD; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (K) Representative ChIP-qPCR of IRF8 and IRF5 binding at the Cxcl cluster in the indicated mice. Data representative of 3 independent experiments and show mean +/− SD; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. *p
    Figure Legend Snippet: IRF8 and IRF5 are required for ABC generation and differentiation in DKO females. (A-C) Quantifications showing the numbers of CD11c + Tbet + ABCs (A) , CD11c + CD11b + ABCs (B) , and total GL7 + Fas + B cells (C) from the spleens of WT, DKO(F), CD23- Irf8 .DKO(F), CD23- Irf5 f/+ .DKO(F), and CD23- Irf5. DKO(F) mice. Data pooled from at least 5 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (D) Representative immunofluorescence images of B220 (blue), GL7 (pink) , and CD3 (green) on the indicated mice. Data representative of at least 4 mice per genotype and show mean +/− SEM; p-value by unpaired two-tailed t-test. (E-F) Quantifications showing the numbers of total PB/PCs (B220 mid/lo CD138 + ) (E) and CD11c + PB/PCs (F) from the indicated mice. Data pooled from at least 5 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (G) ELISA data showing anti-dsDNA IgG2c in the serum from the indicated mice. Data pooled from at least 4 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (H-K) CD23 + B cells from the indicated mice were stimulated for 2-3d with α IgM, α CD40, and IL-21. (H) Quantification of CD11c + Tbet + B cells after 3d culture. Data pooled from at least 3 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (I) Quantifications showing the expression of CD11c and Tbet in the indicated strains after 3d culture. Data pooled from at least 4 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (J) ELISA data showing CXCL10 production in the supernatants after 3d culture. Data representative of 3 independent experiments and show mean +/− SD; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (K) Representative ChIP-qPCR of IRF8 and IRF5 binding at the Cxcl cluster in the indicated mice. Data representative of 3 independent experiments and show mean +/− SD; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. *p

    Techniques Used: Mouse Assay, Immunofluorescence, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    (A) Representative histograms and quantifications of Fcrl5, Tbet, Cxcr3, CD11b, IgM, IgD, and CD44 expression in CD11c + CD19 + GL7 + Fas + cells (CD11c + ) ( red ), CD11c − CD19 + GL7 + Fas + cells (CD11c − ) (black) , and CD19 + CD11c + CD11b + (ABCs) (green) from DKO(F) mice. CD19 + Fas − GL7 − cells are shown as a control (gray) . Data representative of and/or pooled from at least 6 mice and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (B) Plot showing the enrichment of an ABC geneset from SLE patients in CD11c + CD19 + GL7 + CD38 lo cells 15 . (C) Plot showing the enrichment of the HALLMARK_INTERFERON_ALPHA_RESPONSE and the HALLMARK_INTERFERON_GAMMA_RESPONSE genesets in CD11c + CD19 + GL7 + CD38 lo cells. (D) Representative histograms and quantifications of IRF8 and BCL6 in the indicated populations from DKO(F) mice. Data representative of and/or pooled from at least 6 mice and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (E-F) Splenocytes from aged DKO mice were co-cultured for 3hr with Cypher5E-labeled thymocytes following induction of apoptosis with 50μM Dexamethasone. (E) Representative histograms and quantifications showing the percentage of CD11c + and CD11c − CD19 + GL7 + Fas + cells that engulfed apoptotic thymocytes (Cypher5E + ). Data representative of and/or pooled from 4 DKO(F), 1 DKO(M), and 1 YAA-DKO(M) mice; p-value by paired two-tailed t-test. (F) Representative histogram and quantification of MHC-II expression in CD11c + CD19 + GL7 + Fas + B cells that engulfed (red ) or did not engulf (blue) apoptotic thymocytes. Data representative of and/or pooled from 6 mice as in Fig. 5E ; p-value by paired two-tailed t-test.
    Figure Legend Snippet: (A) Representative histograms and quantifications of Fcrl5, Tbet, Cxcr3, CD11b, IgM, IgD, and CD44 expression in CD11c + CD19 + GL7 + Fas + cells (CD11c + ) ( red ), CD11c − CD19 + GL7 + Fas + cells (CD11c − ) (black) , and CD19 + CD11c + CD11b + (ABCs) (green) from DKO(F) mice. CD19 + Fas − GL7 − cells are shown as a control (gray) . Data representative of and/or pooled from at least 6 mice and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (B) Plot showing the enrichment of an ABC geneset from SLE patients in CD11c + CD19 + GL7 + CD38 lo cells 15 . (C) Plot showing the enrichment of the HALLMARK_INTERFERON_ALPHA_RESPONSE and the HALLMARK_INTERFERON_GAMMA_RESPONSE genesets in CD11c + CD19 + GL7 + CD38 lo cells. (D) Representative histograms and quantifications of IRF8 and BCL6 in the indicated populations from DKO(F) mice. Data representative of and/or pooled from at least 6 mice and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (E-F) Splenocytes from aged DKO mice were co-cultured for 3hr with Cypher5E-labeled thymocytes following induction of apoptosis with 50μM Dexamethasone. (E) Representative histograms and quantifications showing the percentage of CD11c + and CD11c − CD19 + GL7 + Fas + cells that engulfed apoptotic thymocytes (Cypher5E + ). Data representative of and/or pooled from 4 DKO(F), 1 DKO(M), and 1 YAA-DKO(M) mice; p-value by paired two-tailed t-test. (F) Representative histogram and quantification of MHC-II expression in CD11c + CD19 + GL7 + Fas + B cells that engulfed (red ) or did not engulf (blue) apoptotic thymocytes. Data representative of and/or pooled from 6 mice as in Fig. 5E ; p-value by paired two-tailed t-test.

    Techniques Used: Expressing, Mouse Assay, Cell Culture, Labeling, Two Tailed Test

    Duplication of Tlr7 in DKO males promotes ABC dissemination and severe immunopathogenesis. (A) Quantification of CD11c + Tbet + ABCs from the spleens, blood, and kidneys of WT, DKO(F), DKO(M), and Yaa-DKO(M) mice. Data shows mean +/− SEM; n > =5 mice per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (B) Glomerulonephritis (GMN) scores of kidneys from the indicated mice. Data shows mean +/− SEM; n > =4 per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (C) Quantification of IgG deposition in the kidneys from the indicated mice. Data shows mean +/− SEM; n > =10 fields across 2-3 mice per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (D) Representative H E images of lungs from the indicated mice. Data representative of 2 WT, 4 DKO(F), 4 DKO(M), and 4 Yaa-DKO(M) mice (30-36wk). (E) Quantifications of the numbers of CD11c + CD11b + ABCs, total GL7 + Fas + B cells, CD11c + GL7 + Fas + B cells, total PB/PCs, and CD11c + PB/PCs in the lungs from the indicated mice. Data show mean +/− SEM; n > = 3 mice per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (F) Plot showing the inflammation score in the lungs of 45wk-old CD23-Cre. Irf8 f/f .DKO(F) and Irf8 f/f .DKO(F) control mice as determined by H E staining. Data shows mean +/− SEM; n =5 per genotype; p-value by unpaired two-tailed t-test. (G) Plots showing the numbers of white blood cells, lymphocytes, and platelets in the blood from the indicated mice. Data show mean +/− SEM; n > =6 per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (H) Luminex data showing the fold-increase in serum levels of TNF α and IL-4 in DKO(F), DKO(M), and Yaa-DKO(M) mice relative to WT. Data pooled from at least 4 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (I) ELISA data for anti- phosphatidylserine (pS) IgM and IgG, anti-Cardiolipin IgG, and anti-MDA-LDL IgG in the serum from the indicated mice. Data shows mean +/− SEM; n > =3 per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (J) RNA-seq was performed on sorted CD11c + PB/PCs (CD138 + TACI + ) from Yaa-DKO(M) mice as in Fig. 6A . Plot shows top pathways enriched in CD11c + PB/PCs from Yaa-DKO(M) mice as compared to CD11c + PB/PCs from DKO(F) mice as determined by GSEA. Dotted line indicates significance threshold at FDR q
    Figure Legend Snippet: Duplication of Tlr7 in DKO males promotes ABC dissemination and severe immunopathogenesis. (A) Quantification of CD11c + Tbet + ABCs from the spleens, blood, and kidneys of WT, DKO(F), DKO(M), and Yaa-DKO(M) mice. Data shows mean +/− SEM; n > =5 mice per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (B) Glomerulonephritis (GMN) scores of kidneys from the indicated mice. Data shows mean +/− SEM; n > =4 per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (C) Quantification of IgG deposition in the kidneys from the indicated mice. Data shows mean +/− SEM; n > =10 fields across 2-3 mice per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (D) Representative H E images of lungs from the indicated mice. Data representative of 2 WT, 4 DKO(F), 4 DKO(M), and 4 Yaa-DKO(M) mice (30-36wk). (E) Quantifications of the numbers of CD11c + CD11b + ABCs, total GL7 + Fas + B cells, CD11c + GL7 + Fas + B cells, total PB/PCs, and CD11c + PB/PCs in the lungs from the indicated mice. Data show mean +/− SEM; n > = 3 mice per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (F) Plot showing the inflammation score in the lungs of 45wk-old CD23-Cre. Irf8 f/f .DKO(F) and Irf8 f/f .DKO(F) control mice as determined by H E staining. Data shows mean +/− SEM; n =5 per genotype; p-value by unpaired two-tailed t-test. (G) Plots showing the numbers of white blood cells, lymphocytes, and platelets in the blood from the indicated mice. Data show mean +/− SEM; n > =6 per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (H) Luminex data showing the fold-increase in serum levels of TNF α and IL-4 in DKO(F), DKO(M), and Yaa-DKO(M) mice relative to WT. Data pooled from at least 4 mice per genotype and show mean +/− SEM; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (I) ELISA data for anti- phosphatidylserine (pS) IgM and IgG, anti-Cardiolipin IgG, and anti-MDA-LDL IgG in the serum from the indicated mice. Data shows mean +/− SEM; n > =3 per genotype; p-value by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (J) RNA-seq was performed on sorted CD11c + PB/PCs (CD138 + TACI + ) from Yaa-DKO(M) mice as in Fig. 6A . Plot shows top pathways enriched in CD11c + PB/PCs from Yaa-DKO(M) mice as compared to CD11c + PB/PCs from DKO(F) mice as determined by GSEA. Dotted line indicates significance threshold at FDR q

    Techniques Used: Mouse Assay, Staining, Two Tailed Test, Luminex, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, RNA Sequencing Assay

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    Staining:

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    Article Snippet: On D7 mice were sacrificed and blood, peritoneal lavage and spleens harvested. .. Splenic CD19+ve B cells were FACs sorted into IL-10-GFP+ve or −ve fractions, phenotyped and cultured for 10 days in the presence of MegaAPRIL [Adipogen (200 ng/ml)], CpGB [ODN1826 Eurofins MWG Operon (1 μg/ml)], and IL-4 [R & D System (50 ng/ml)], after which culture supernatants were checked for IgM and IgG levels (Ready-SET-Go ELISA kit eBioScience). .. Highly purified IL-10-GFP+ve B1a cells, generated in vivo , were fused with SP2/0 cells to generate hybridomas.

    Article Title: Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells
    Article Snippet: For cell activation experiments, B cells were extrinsically sialylated with 40 μg/ml ST6Gal-1 and 0.05 mM CMP-sialic acid (Sigma C-8271) in serum-free RPMI for 2 h, then stimulated with 200 ng/ml murine BAFF (R & D Biosystems) or 10 μg/ml function-grade anti-IgM F(ab')2 (Invitrogen 16-5092-85). .. To model negative selection, cells were cultured at 1 × 105 cells/ml as indicated in presence of 10 μg/ml ST6Gal-1, 0.05 mM CMP-sialic acid, 20 ng/ml BAFF, 10 μg/ml anti-IgM antibody, 1000 U/ml IL-4, and function-grade anti-CD40 antibody (eBioscience HM40-3) for 18–20 h. Live cells were quantified by DAPI flow cytometry. .. Recombinant rat secretory ST6Gal-1 was a generous gift from Dr. Kelley Moremen of the University of Georgia.

    Selection:

    Article Title: Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells
    Article Snippet: For cell activation experiments, B cells were extrinsically sialylated with 40 μg/ml ST6Gal-1 and 0.05 mM CMP-sialic acid (Sigma C-8271) in serum-free RPMI for 2 h, then stimulated with 200 ng/ml murine BAFF (R & D Biosystems) or 10 μg/ml function-grade anti-IgM F(ab')2 (Invitrogen 16-5092-85). .. To model negative selection, cells were cultured at 1 × 105 cells/ml as indicated in presence of 10 μg/ml ST6Gal-1, 0.05 mM CMP-sialic acid, 20 ng/ml BAFF, 10 μg/ml anti-IgM antibody, 1000 U/ml IL-4, and function-grade anti-CD40 antibody (eBioscience HM40-3) for 18–20 h. Live cells were quantified by DAPI flow cytometry. .. Recombinant rat secretory ST6Gal-1 was a generous gift from Dr. Kelley Moremen of the University of Georgia.

    Flow Cytometry:

    Article Title: Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells
    Article Snippet: For cell activation experiments, B cells were extrinsically sialylated with 40 μg/ml ST6Gal-1 and 0.05 mM CMP-sialic acid (Sigma C-8271) in serum-free RPMI for 2 h, then stimulated with 200 ng/ml murine BAFF (R & D Biosystems) or 10 μg/ml function-grade anti-IgM F(ab')2 (Invitrogen 16-5092-85). .. To model negative selection, cells were cultured at 1 × 105 cells/ml as indicated in presence of 10 μg/ml ST6Gal-1, 0.05 mM CMP-sialic acid, 20 ng/ml BAFF, 10 μg/ml anti-IgM antibody, 1000 U/ml IL-4, and function-grade anti-CD40 antibody (eBioscience HM40-3) for 18–20 h. Live cells were quantified by DAPI flow cytometry. .. Recombinant rat secretory ST6Gal-1 was a generous gift from Dr. Kelley Moremen of the University of Georgia.

    Cytometry:

    Article Title: Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells
    Article Snippet: For cell activation experiments, B cells were extrinsically sialylated with 40 μg/ml ST6Gal-1 and 0.05 mM CMP-sialic acid (Sigma C-8271) in serum-free RPMI for 2 h, then stimulated with 200 ng/ml murine BAFF (R & D Biosystems) or 10 μg/ml function-grade anti-IgM F(ab')2 (Invitrogen 16-5092-85). .. To model negative selection, cells were cultured at 1 × 105 cells/ml as indicated in presence of 10 μg/ml ST6Gal-1, 0.05 mM CMP-sialic acid, 20 ng/ml BAFF, 10 μg/ml anti-IgM antibody, 1000 U/ml IL-4, and function-grade anti-CD40 antibody (eBioscience HM40-3) for 18–20 h. Live cells were quantified by DAPI flow cytometry. .. Recombinant rat secretory ST6Gal-1 was a generous gift from Dr. Kelley Moremen of the University of Georgia.

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    Thermo Fisher mouse anti human pd 1 antibody
    <t>PD-1</t> blockade decreases Bim expression induced by PD-L1 in human CD8 + T cells.
    Mouse Anti Human Pd 1 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti human tnf
    <t>TNF</t> and <t>IFN-β</t> are required for potent restriction of MV infection in primary human fibroblasts. (A) Primary human skin fibroblasts CCD-922Sk were mock-infected or infected with MV. X-gal staining was performed to visualize blue MV foci 48 h after infection. (B) CCD-922Sk fibroblasts were infected with MV in the absence or presence of TNF or IFN-β alone or TNF plus IFN-β. MV yields were determined by the standard plaque assay using BGMK cells at 48 h after infection. (C) CCD-922Sk fibroblasts were infected with the control MV (constructed with the same vector but no human TNF gene) or the recombinant MV expressing human TNF (MV-hTNF) in the absence or presence of IFN-β. The viral yields of both control MV and MV-hTNF were determined by the standard plaque assay using RK-13 cells at 48 h after infection. (D) CCD-922Sk fibroblasts were infected with EMCV in the absence or presence of IFN-β or TNF. EMCV quantities were titrated by the standard plaque assay using BHK cells at 48 h after infection. Data in (B), (C) and (D) represent mean +/− SD.
    Anti Human Tnf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gold conjugated goat anti mouse antibody
    Localization of NADPH oxidase proteins in rabbit aorta. Aortic sections were incubated with control nonimmune <t>mouse</t> serum ( A ) or monoclonal antibodies against p22 phox (1:100) ( B and C ), gp91 phox (1:200) ( D ), p47 phox (1:20) ( E ), and p67 phox (1:20) ( F ) at the dilutions shown, followed by <t>gold-conjugated</t> <t>goat</t> <t>anti-mouse</t> antibody. The sections were developed as described in Methods . Magnification is 190× ( A and C–F ) and 95× ( B ). The sections shown represent at least three stained sections for each antibody.
    Gold Conjugated Goat Anti Mouse Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher monoclonal anti human immunoglobulin m igm
    Western blot of P. knowlesi pooled sera probed with P. vivax pooled sera. Unfractionated, pooled serum samples from patient is subjected to 2-DE, transferred onto nitrocellulose membranes, and probed with pooled sera (as primary antibody) followed by monoclonal anti-human <t>IgM-HRP</t> (as secondary antibody).
    Monoclonal Anti Human Immunoglobulin M Igm, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PD-1 blockade decreases Bim expression induced by PD-L1 in human CD8 + T cells.

    Journal: JCI Insight

    Article Title: T cell Bim levels reflect responses to anti–PD-1 cancer therapy

    doi: 10.1172/jci.insight.86014

    Figure Lengend Snippet: PD-1 blockade decreases Bim expression induced by PD-L1 in human CD8 + T cells.

    Article Snippet: To block PD-1/PD-L1 interaction-induced Bim upregulation, preactivated CD8+ T cells were incubated with 10 μg/ml of mouse anti-human PD-1 antibody (clone MIH4, eBioscience, special order for functional grade-purified version) or humanized antibody to PD-1 (Keytruda or Opdivo, Mayo Clinic Pharmacy) for 20 minutes before culture with PD-L1 fusion protein.

    Techniques: Expressing

    Bim upregulation is associated with PD-1 expression in melanoma patients.

    Journal: JCI Insight

    Article Title: T cell Bim levels reflect responses to anti–PD-1 cancer therapy

    doi: 10.1172/jci.insight.86014

    Figure Lengend Snippet: Bim upregulation is associated with PD-1 expression in melanoma patients.

    Article Snippet: To block PD-1/PD-L1 interaction-induced Bim upregulation, preactivated CD8+ T cells were incubated with 10 μg/ml of mouse anti-human PD-1 antibody (clone MIH4, eBioscience, special order for functional grade-purified version) or humanized antibody to PD-1 (Keytruda or Opdivo, Mayo Clinic Pharmacy) for 20 minutes before culture with PD-L1 fusion protein.

    Techniques: Expressing

    Changes of Bim levels predict responses to anti–PD-1 therapy in melanoma patients.

    Journal: JCI Insight

    Article Title: T cell Bim levels reflect responses to anti–PD-1 cancer therapy

    doi: 10.1172/jci.insight.86014

    Figure Lengend Snippet: Changes of Bim levels predict responses to anti–PD-1 therapy in melanoma patients.

    Article Snippet: To block PD-1/PD-L1 interaction-induced Bim upregulation, preactivated CD8+ T cells were incubated with 10 μg/ml of mouse anti-human PD-1 antibody (clone MIH4, eBioscience, special order for functional grade-purified version) or humanized antibody to PD-1 (Keytruda or Opdivo, Mayo Clinic Pharmacy) for 20 minutes before culture with PD-L1 fusion protein.

    Techniques:

    Bim upregulation in tumor-reactive CD8 + T cells is dependent on PD-1 and PD-L1 interaction.

    Journal: JCI Insight

    Article Title: T cell Bim levels reflect responses to anti–PD-1 cancer therapy

    doi: 10.1172/jci.insight.86014

    Figure Lengend Snippet: Bim upregulation in tumor-reactive CD8 + T cells is dependent on PD-1 and PD-L1 interaction.

    Article Snippet: To block PD-1/PD-L1 interaction-induced Bim upregulation, preactivated CD8+ T cells were incubated with 10 μg/ml of mouse anti-human PD-1 antibody (clone MIH4, eBioscience, special order for functional grade-purified version) or humanized antibody to PD-1 (Keytruda or Opdivo, Mayo Clinic Pharmacy) for 20 minutes before culture with PD-L1 fusion protein.

    Techniques:

    TNF and IFN-β are required for potent restriction of MV infection in primary human fibroblasts. (A) Primary human skin fibroblasts CCD-922Sk were mock-infected or infected with MV. X-gal staining was performed to visualize blue MV foci 48 h after infection. (B) CCD-922Sk fibroblasts were infected with MV in the absence or presence of TNF or IFN-β alone or TNF plus IFN-β. MV yields were determined by the standard plaque assay using BGMK cells at 48 h after infection. (C) CCD-922Sk fibroblasts were infected with the control MV (constructed with the same vector but no human TNF gene) or the recombinant MV expressing human TNF (MV-hTNF) in the absence or presence of IFN-β. The viral yields of both control MV and MV-hTNF were determined by the standard plaque assay using RK-13 cells at 48 h after infection. (D) CCD-922Sk fibroblasts were infected with EMCV in the absence or presence of IFN-β or TNF. EMCV quantities were titrated by the standard plaque assay using BHK cells at 48 h after infection. Data in (B), (C) and (D) represent mean +/− SD.

    Journal: PLoS Pathogens

    Article Title: RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

    doi: 10.1371/journal.ppat.1000099

    Figure Lengend Snippet: TNF and IFN-β are required for potent restriction of MV infection in primary human fibroblasts. (A) Primary human skin fibroblasts CCD-922Sk were mock-infected or infected with MV. X-gal staining was performed to visualize blue MV foci 48 h after infection. (B) CCD-922Sk fibroblasts were infected with MV in the absence or presence of TNF or IFN-β alone or TNF plus IFN-β. MV yields were determined by the standard plaque assay using BGMK cells at 48 h after infection. (C) CCD-922Sk fibroblasts were infected with the control MV (constructed with the same vector but no human TNF gene) or the recombinant MV expressing human TNF (MV-hTNF) in the absence or presence of IFN-β. The viral yields of both control MV and MV-hTNF were determined by the standard plaque assay using RK-13 cells at 48 h after infection. (D) CCD-922Sk fibroblasts were infected with EMCV in the absence or presence of IFN-β or TNF. EMCV quantities were titrated by the standard plaque assay using BHK cells at 48 h after infection. Data in (B), (C) and (D) represent mean +/− SD.

    Article Snippet: The primary antibodies were obtained from the following suppliers: anti-IRF3, anti-IRF7, anti-NF-κB p65 and anti-USF2 antibodies, Santa Cruz Biotechnology; anti-human TNF and anti-human IFN-α/β antibodies, Biosource International; anti-β-actin antibody, Sigma; anti-STAT1 antibody, Transduction Laboratories.

    Techniques: Infection, Staining, Plaque Assay, Construct, Plasmid Preparation, Recombinant, Expressing

    MV-elicited production of TNF and IFN-β is mediated by RIG-I in primary human macrophages. (A) pHMs were transfected with control siRNA or siRNAs targeting cytoplasmic RNA sensors RIG-I (top panel) or MDA5 (middle panel). The cells were analyzed 48 h later by RT-PCR for the indicated mRNA levels. GAPDH was used as control. (B, C) RIG-I is critically required for MV-elicited production of both TNF (B) and IFN-β (C) in pHMs. pHMs or various siRNA pHMs were mock-infected or infected with MV and the accumulation of TNF and IFN-β in the culture supernatants was assessed by ELISA 24 h post-infection. (D) RIG-I mediates cellular restriction to MV infection in pHMs. pHMs or control siRNA or RIG-I siRNA pHMs as indicated were infected with MV. MV yields were determined by the standard plaque assay using BGMK cells at 48 h after infection. Data in (B), (C) and (D) represent mean +/− SD.

    Journal: PLoS Pathogens

    Article Title: RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

    doi: 10.1371/journal.ppat.1000099

    Figure Lengend Snippet: MV-elicited production of TNF and IFN-β is mediated by RIG-I in primary human macrophages. (A) pHMs were transfected with control siRNA or siRNAs targeting cytoplasmic RNA sensors RIG-I (top panel) or MDA5 (middle panel). The cells were analyzed 48 h later by RT-PCR for the indicated mRNA levels. GAPDH was used as control. (B, C) RIG-I is critically required for MV-elicited production of both TNF (B) and IFN-β (C) in pHMs. pHMs or various siRNA pHMs were mock-infected or infected with MV and the accumulation of TNF and IFN-β in the culture supernatants was assessed by ELISA 24 h post-infection. (D) RIG-I mediates cellular restriction to MV infection in pHMs. pHMs or control siRNA or RIG-I siRNA pHMs as indicated were infected with MV. MV yields were determined by the standard plaque assay using BGMK cells at 48 h after infection. Data in (B), (C) and (D) represent mean +/− SD.

    Article Snippet: The primary antibodies were obtained from the following suppliers: anti-IRF3, anti-IRF7, anti-NF-κB p65 and anti-USF2 antibodies, Santa Cruz Biotechnology; anti-human TNF and anti-human IFN-α/β antibodies, Biosource International; anti-β-actin antibody, Sigma; anti-STAT1 antibody, Transduction Laboratories.

    Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Infection, Enzyme-linked Immunosorbent Assay, Plaque Assay

    Primary human macrophages produce TNF and IFN-β in response to MV infection. (A, B) Primary human macrophages (pHMs), CCD-922Sk fibroblasts and primary human lymphocytes were mock-infected or infected with MV. TNF (A) and IFN-β (B) accumulation in the culture supernatants was assessed by ELISA 24 h post-infection. (C) pHMs were mock-infected or infected with MV in the absence or presence of TNF neutralizing antibody (Ab) or IFN-α/β neutralizing Ab alone or TNF Ab plus IFN-α/β Ab as specified. MV β-gal activity was determined by measuring absorbance at 420 nm and normalized to the total cellular protein levels at 48 h after infection. (D) pHMs were infected with MV or UV-inactivated MV for various times (below lanes) and total RNA was analyzed by RT-PCR for induction of TNF and IFN-β mRNA. GAPDH was used as control. Data in (A), (B) and (C) represent mean +/− SD.

    Journal: PLoS Pathogens

    Article Title: RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

    doi: 10.1371/journal.ppat.1000099

    Figure Lengend Snippet: Primary human macrophages produce TNF and IFN-β in response to MV infection. (A, B) Primary human macrophages (pHMs), CCD-922Sk fibroblasts and primary human lymphocytes were mock-infected or infected with MV. TNF (A) and IFN-β (B) accumulation in the culture supernatants was assessed by ELISA 24 h post-infection. (C) pHMs were mock-infected or infected with MV in the absence or presence of TNF neutralizing antibody (Ab) or IFN-α/β neutralizing Ab alone or TNF Ab plus IFN-α/β Ab as specified. MV β-gal activity was determined by measuring absorbance at 420 nm and normalized to the total cellular protein levels at 48 h after infection. (D) pHMs were infected with MV or UV-inactivated MV for various times (below lanes) and total RNA was analyzed by RT-PCR for induction of TNF and IFN-β mRNA. GAPDH was used as control. Data in (A), (B) and (C) represent mean +/− SD.

    Article Snippet: The primary antibodies were obtained from the following suppliers: anti-IRF3, anti-IRF7, anti-NF-κB p65 and anti-USF2 antibodies, Santa Cruz Biotechnology; anti-human TNF and anti-human IFN-α/β antibodies, Biosource International; anti-β-actin antibody, Sigma; anti-STAT1 antibody, Transduction Laboratories.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    IRF7 is crucial for sustaining MV-elicited TNF induction in primary human macrophages. (A) Control siRNA pHMs or IRF7 siRNA pHMs were infected with MV for various times (below lanes) and the whole cell lysates were analyzed for MV-induced expression of IRF7 protein. β-actin was used as control. (B) pHMs or various siRNA pHMs were mock-infected or infected with MV. TNF protein in the culture supernatants was assessed by ELISA 24 h post-infection. (C) IRF7 expression and TNF production kinetics. Control siRNA pHMs or IRF7 siRNA pHMs were infected with MV for various times as indicated and TNF protein in the culture supernatants was assessed by ELISA. (D) IRF7 is required for sustaining TNF gene transcription activated by MV infection. Control siRNA pHMs or IRF7 siRNA pHMs were infected with MV for various times (below lanes) and the cells were analyzed by RT-PCR for the indicated TNF mRNA levels. GAPDH was used as control. Data in (B) and (C) represent mean +/− SD.

    Journal: PLoS Pathogens

    Article Title: RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

    doi: 10.1371/journal.ppat.1000099

    Figure Lengend Snippet: IRF7 is crucial for sustaining MV-elicited TNF induction in primary human macrophages. (A) Control siRNA pHMs or IRF7 siRNA pHMs were infected with MV for various times (below lanes) and the whole cell lysates were analyzed for MV-induced expression of IRF7 protein. β-actin was used as control. (B) pHMs or various siRNA pHMs were mock-infected or infected with MV. TNF protein in the culture supernatants was assessed by ELISA 24 h post-infection. (C) IRF7 expression and TNF production kinetics. Control siRNA pHMs or IRF7 siRNA pHMs were infected with MV for various times as indicated and TNF protein in the culture supernatants was assessed by ELISA. (D) IRF7 is required for sustaining TNF gene transcription activated by MV infection. Control siRNA pHMs or IRF7 siRNA pHMs were infected with MV for various times (below lanes) and the cells were analyzed by RT-PCR for the indicated TNF mRNA levels. GAPDH was used as control. Data in (B) and (C) represent mean +/− SD.

    Article Snippet: The primary antibodies were obtained from the following suppliers: anti-IRF3, anti-IRF7, anti-NF-κB p65 and anti-USF2 antibodies, Santa Cruz Biotechnology; anti-human TNF and anti-human IFN-α/β antibodies, Biosource International; anti-β-actin antibody, Sigma; anti-STAT1 antibody, Transduction Laboratories.

    Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

    IRF3 is critical for triggering MV-elicited TNF induction in primary human macrophages. (A) pHMs were transfected with control siRNA or MAVS siRNA. The cells were analyzed 48 h later by RT-PCR for the indicated mRNA levels. GAPDH was used as the control. (B) Control siRNA pHMs or RIG-I siRNA or MAVS siRNA pHMs as indicated were infected with MV for various times (below lanes) and the nuclear extracts were probed for nuclear IRF3 (nuc-IRF3). Nuclear USF2 (nuc-USF2) was used as nuclear protein loading control. (C) Control siRNA pHMs or RIG-I siRNA or MAVS siRNA pHMs as indicated were infected with MV for 0 h or 10 h and were immunofluorescence-stained for IRF3 (red). Nuclear DNA was counterstained with DAPI (blue). Bars are 10 µm. (D) pHMs were transfected with control siRNA or IRF3 siRNA as indicated and were analyzed 72 h later by immunoblotting for IRF3 protein levels. β-actin was used as control. (E) pHMs or various siRNA pHMs as indicated were infected with MV for 24 h and TNF protein in the culture supernatants was assessed by ELISA. Data represent mean +/− SD. (F) Control siRNA pHMs or IRF3 siRNA pHMs were infected with MV for various times (below lanes) and the cells were analyzed by RT-PCR for the indicated TNF mRNA levels. GAPDH was used as control.

    Journal: PLoS Pathogens

    Article Title: RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

    doi: 10.1371/journal.ppat.1000099

    Figure Lengend Snippet: IRF3 is critical for triggering MV-elicited TNF induction in primary human macrophages. (A) pHMs were transfected with control siRNA or MAVS siRNA. The cells were analyzed 48 h later by RT-PCR for the indicated mRNA levels. GAPDH was used as the control. (B) Control siRNA pHMs or RIG-I siRNA or MAVS siRNA pHMs as indicated were infected with MV for various times (below lanes) and the nuclear extracts were probed for nuclear IRF3 (nuc-IRF3). Nuclear USF2 (nuc-USF2) was used as nuclear protein loading control. (C) Control siRNA pHMs or RIG-I siRNA or MAVS siRNA pHMs as indicated were infected with MV for 0 h or 10 h and were immunofluorescence-stained for IRF3 (red). Nuclear DNA was counterstained with DAPI (blue). Bars are 10 µm. (D) pHMs were transfected with control siRNA or IRF3 siRNA as indicated and were analyzed 72 h later by immunoblotting for IRF3 protein levels. β-actin was used as control. (E) pHMs or various siRNA pHMs as indicated were infected with MV for 24 h and TNF protein in the culture supernatants was assessed by ELISA. Data represent mean +/− SD. (F) Control siRNA pHMs or IRF3 siRNA pHMs were infected with MV for various times (below lanes) and the cells were analyzed by RT-PCR for the indicated TNF mRNA levels. GAPDH was used as control.

    Article Snippet: The primary antibodies were obtained from the following suppliers: anti-IRF3, anti-IRF7, anti-NF-κB p65 and anti-USF2 antibodies, Santa Cruz Biotechnology; anti-human TNF and anti-human IFN-α/β antibodies, Biosource International; anti-β-actin antibody, Sigma; anti-STAT1 antibody, Transduction Laboratories.

    Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Infection, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

    Localization of NADPH oxidase proteins in rabbit aorta. Aortic sections were incubated with control nonimmune mouse serum ( A ) or monoclonal antibodies against p22 phox (1:100) ( B and C ), gp91 phox (1:200) ( D ), p47 phox (1:20) ( E ), and p67 phox (1:20) ( F ) at the dilutions shown, followed by gold-conjugated goat anti-mouse antibody. The sections were developed as described in Methods . Magnification is 190× ( A and C–F ) and 95× ( B ). The sections shown represent at least three stained sections for each antibody.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: Enhancement by angiotensin II

    doi:

    Figure Lengend Snippet: Localization of NADPH oxidase proteins in rabbit aorta. Aortic sections were incubated with control nonimmune mouse serum ( A ) or monoclonal antibodies against p22 phox (1:100) ( B and C ), gp91 phox (1:200) ( D ), p47 phox (1:20) ( E ), and p67 phox (1:20) ( F ) at the dilutions shown, followed by gold-conjugated goat anti-mouse antibody. The sections were developed as described in Methods . Magnification is 190× ( A and C–F ) and 95× ( B ). The sections shown represent at least three stained sections for each antibody.

    Article Snippet: After washing with blotto, the sections were incubated for 60 min at 25°C with 5 nm gold-conjugated goat anti-mouse antibody (Zymed) diluted 1:50 in the same buffer.

    Techniques: Incubation, Staining

    Western blot of P. knowlesi pooled sera probed with P. vivax pooled sera. Unfractionated, pooled serum samples from patient is subjected to 2-DE, transferred onto nitrocellulose membranes, and probed with pooled sera (as primary antibody) followed by monoclonal anti-human IgM-HRP (as secondary antibody).

    Journal: BMC Infectious Diseases

    Article Title: Identification of circulating biomarkers in sera of Plasmodium knowlesi-infected malaria patients – comparison against Plasmodium vivax infection

    doi: 10.1186/s12879-015-0786-2

    Figure Lengend Snippet: Western blot of P. knowlesi pooled sera probed with P. vivax pooled sera. Unfractionated, pooled serum samples from patient is subjected to 2-DE, transferred onto nitrocellulose membranes, and probed with pooled sera (as primary antibody) followed by monoclonal anti-human IgM-HRP (as secondary antibody).

    Article Snippet: After another washing step, the membranes were incubated with monoclonal anti-human Immunoglobulin M (IgM) conjugated to horseradish peroxidase (HRP) (Invitrogen, USA) at a dilution of 1:6,000 for 1 h at room temperature.

    Techniques: Western Blot

    Western blot results following 2-DE. (a) normal pooled sera probed with normal pooled sera, (b) normal pooled sera probed with P. knowlesi pooled sera, (c) P. knowlesi pooled sera probed with normal pooled sera, (d) P. knowlesi pooled sera probed with P. knowlesi pooled sera, Unfractionated, pooled serum samples from patients and normal controls were subjected to 2-DE, transferred onto nitrocellulose membranes, and probed with pooled sera (as primary antibody) followed by monoclonal anti-human IgM-HRP (as secondary antibody).

    Journal: BMC Infectious Diseases

    Article Title: Identification of circulating biomarkers in sera of Plasmodium knowlesi-infected malaria patients – comparison against Plasmodium vivax infection

    doi: 10.1186/s12879-015-0786-2

    Figure Lengend Snippet: Western blot results following 2-DE. (a) normal pooled sera probed with normal pooled sera, (b) normal pooled sera probed with P. knowlesi pooled sera, (c) P. knowlesi pooled sera probed with normal pooled sera, (d) P. knowlesi pooled sera probed with P. knowlesi pooled sera, Unfractionated, pooled serum samples from patients and normal controls were subjected to 2-DE, transferred onto nitrocellulose membranes, and probed with pooled sera (as primary antibody) followed by monoclonal anti-human IgM-HRP (as secondary antibody).

    Article Snippet: After another washing step, the membranes were incubated with monoclonal anti-human Immunoglobulin M (IgM) conjugated to horseradish peroxidase (HRP) (Invitrogen, USA) at a dilution of 1:6,000 for 1 h at room temperature.

    Techniques: Western Blot