Journal: iScience

Article Title: Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption

doi: 10.1016/j.isci.2023.107580

Figure Lengend Snippet: Serum levels of total and HDM-specific immunoglobulins show a time-of-day response and sex-based differences to chronic HDM exposure Total IgE, Total IgG, HDM-specific IgE, HDM-specific IgG, HDM-specific IgG1, HDM-specific IgG2b, HDM-specific IgM, and HDM-specific IgA in the serum of chronic PBS- and HDM-exposed females and males at ZT0 and ZT12 were determined by ELISA. Data were expressed as ng/ml and mg/ml for total IgE and total IgG, respectively. HDM-specific immunoglobulins (IgE, IgG, IgG1, IgG2b, IgM, and IgA) in the serum were determined by commercially available ELISA kits (Chondrex, Inc.). Unable to detect HDM-specific IgG2a and IgG3 levels in PBS- and HDM-exposed mice. Data for the HDM-specific immunoglobulins were expressed as absorbance at 450 nm or μg/ml. Data are shown as mean ± SEM, Two-way ANOVA followed by Tukey’s multiple comparison test (n = 5/group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, compared to respective control (PBS) at ZT0 or ZT12; # p < 0.05, compared to HDM at ZT0 vs. ZT12; # # p < 0.01, compared to HDM at ZT0 vs. ZT12. Summary statistics for interaction between Treatment x Sex x Time were analyzed using generalized linear modeling using R (see Table S2 ).

Article Snippet: Mouse Anti-House Dust Mite (HDM) IgG2b Antibody Assay Kit , Chondrex, Inc. , Cat# 3035.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: iScience

Article Title: Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption

doi: 10.1016/j.isci.2023.107580

Figure Lengend Snippet:

Article Snippet: Mouse Anti-House Dust Mite (HDM) IgG2b Antibody Assay Kit , Chondrex, Inc. , Cat# 3035.

Techniques: Purification, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Software

Journal: Scientific Reports

Article Title: Natriuretic Peptide Receptor B modulates the proliferation of the cardiac cells expressing the Stem Cell Antigen-1

doi: 10.1038/srep41936

Figure Lengend Snippet: ( A ) Non myocyte cells (NMCs) were isolated from neonatal hearts of C57BL/6 mice, cultured 4 and 11 days with or without BNP (untreated cells) and counted. ( B ) Percentages of c-kit + and Sca-1 + cells obtained by flow cytometry analysis on BNP treated or untreated NMCs. ( C ) Number of cells expressing the c-kit or the Sca-1 protein in NMCs treated or not with BNP for 4 and 11 days calculated with the total number of cells and the percentages of the c-kit + and Sca-1 + cells. ( A – C ) n = 8 and 16 different experiments after 4 and 11 days of culture, respectively. ( D ) Representative histogram of NMC sorting for Sca-1 expression. The numbers represent the percentage of the cells compared to the total number of sorted NMCs. n = 18–43 different experiments. ( E ) Number of sorted Sca-1 − and Sca-1 high+ cells treated or not with BNP for 11 days. n = 6 and 12 for Sca-1 − and Sca-1 high+ cells, respectively. ( F ) Percentages of Sca-1 + cells among sorted Sca-1 − cells treated or not with BNP for 9 days. n = 4 different experiments. ( A – E ) All results expressed as fold-increase above the results obtained in untreated cells. All the results are means ± SEM. *p < 0.05.

Article Snippet: Thus, after isolation, at confluence, the cells were diluted 1:3 before to be either sorted for their Sca-1 expression or directly treated with BNP (1 microM murine BNP (recombinant mouse C 1–45 peptide; American Peptide Co)), CNP (1 microM C-type Natriuretic Peptide (recombinant CNP-22 peptide; Phoenix Pharmaceuticals, Inc)) or ANP (1 microM, Phoenix Pharmaceuticals, Inc).

Techniques: Isolation, Cell Culture, Flow Cytometry, Expressing

Journal: Scientific Reports

Article Title: Natriuretic Peptide Receptor B modulates the proliferation of the cardiac cells expressing the Stem Cell Antigen-1

doi: 10.1038/srep41936

Figure Lengend Snippet: ( A ) Representative immunostainings of sorted Sca-1 high+ cells treated or not with BNP for up to 11 days and stained with antibodies against Nkx2.5 (pink), Proliferating Cell Nuclear Antigen (PCNA) (green) and DAPI (blue). White arrows represent proliferating Nkx2.5 + cells. Scale bars represent 100 μm. ( B ) Quantification of the number of Nkx2.5 + cells among Sca-1 high+ cells treated or not with BNP for up to 11 days. The number of Nkx2.5 + cells was related to the total number of nuclei stained with DAPI. n = 5–6 different experiments and in total 3607 cells in untreated and 3867 cells in BNP treated cells were evaluated for the Nkx2.5 expression. ( C ) Quantitative polymerase chain reaction in untreated (n = 13 different experiments) and BNP treated Sca-1 high+ sorted cells (n = 14). Results are expressed as 2 −ΔΔCT with untreated cells as reference. ( B and C ) Results expressed as fold-increase above the results in untreated cells. All the results are means ± SEM. *p < 0.05.

Article Snippet: Thus, after isolation, at confluence, the cells were diluted 1:3 before to be either sorted for their Sca-1 expression or directly treated with BNP (1 microM murine BNP (recombinant mouse C 1–45 peptide; American Peptide Co)), CNP (1 microM C-type Natriuretic Peptide (recombinant CNP-22 peptide; Phoenix Pharmaceuticals, Inc)) or ANP (1 microM, Phoenix Pharmaceuticals, Inc).

Techniques: Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Natriuretic Peptide Receptor B modulates the proliferation of the cardiac cells expressing the Stem Cell Antigen-1

doi: 10.1038/srep41936

Figure Lengend Snippet: ( A ) Non-myocyte cells (NMCs) isolated from the hearts of neonatal NPR-A or NPR-B deficient mice were cultured with and without BNP. The total number of cells, the percentages of Sca-1 + cells obtained by flow cytometry analysis as well as the number of Sca-1 + cells were determined 11 days after the onset of BNP treatment. n = 9 different experiments for NPR-A and NPR-B deficient cells. ( B and D ) Sorted Sca-1 high+ cells isolated from wild type hearts were cultured with BNP or CNP in presence or not of NPR-B receptor antagonist (P19, 0.5–1 microM) and of an ANF antagonist (Anantin, 0.2 microM). The number of cells was counted after 9–11 days of culture and the results were related to untreated cells. n = at least 4 different experiments. ( C ) NMCs isolated from wild type hearts were treated with C-natriuretic peptide (CNP) for up to 11 days. The total number of cells as well as the number of c-kit + and Sca-1 + cells were determined after counting and flow cytometry analysis. n = 6 different experiments ( D ). ( A – D ) Results expressed as fold-increase above the results in the related untreated cells. All the results are means ± SEM. *p < 0.05 versus the related untreated cells, ° p < 0.05 versus the untreated cells without inhibitors, § p < 0.05 versus the BNP treated cells without inhibitors.

Article Snippet: Thus, after isolation, at confluence, the cells were diluted 1:3 before to be either sorted for their Sca-1 expression or directly treated with BNP (1 microM murine BNP (recombinant mouse C 1–45 peptide; American Peptide Co)), CNP (1 microM C-type Natriuretic Peptide (recombinant CNP-22 peptide; Phoenix Pharmaceuticals, Inc)) or ANP (1 microM, Phoenix Pharmaceuticals, Inc).

Techniques: Isolation, Cell Culture, Flow Cytometry

Journal: Scientific Reports

Article Title: Natriuretic Peptide Receptor B modulates the proliferation of the cardiac cells expressing the Stem Cell Antigen-1

doi: 10.1038/srep41936

Figure Lengend Snippet: ( A ) cGMP levels were measured in the non myocyte cell supernatants (n = 4–7) and in the EDTA treated plasma of mice (n = 4 mice per group) 1h after BNP or CNP treatment (1 μM). ( B ) Representative western blots of sorted Sca-1 high+ cells isolated from B6 + neonatal hearts and stimulated with or without BNP or CNP (1 μM both). Blots were stained with antibodies against phospho phospholamban (pPLB), phospholamban (PBL), phospho p38 (pp38), p38 and Tubulin (used as loading control). Only the bands at the adequate molecular weight were represented here: tubulin 55 kDa, pp38 and p38 about 43 kDa, pPLB between 21 and 26 kDa and PLB 25 kDa. ( C ) Quantification of the data from western blot analysis expressed relatively to the average of untreated cells. Sorted Sca-1 high+ cells were or not treated 1h with BNP or CNP. n = 4–6 different experiments. All the results are means ± SEM, *p < 0.05 versus untreated cells.

Article Snippet: Thus, after isolation, at confluence, the cells were diluted 1:3 before to be either sorted for their Sca-1 expression or directly treated with BNP (1 microM murine BNP (recombinant mouse C 1–45 peptide; American Peptide Co)), CNP (1 microM C-type Natriuretic Peptide (recombinant CNP-22 peptide; Phoenix Pharmaceuticals, Inc)) or ANP (1 microM, Phoenix Pharmaceuticals, Inc).

Techniques: Clinical Proteomics, Western Blot, Isolation, Staining, Control, Molecular Weight

Journal: Scientific Reports

Article Title: Natriuretic Peptide Receptor B modulates the proliferation of the cardiac cells expressing the Stem Cell Antigen-1

doi: 10.1038/srep41936

Figure Lengend Snippet: ( A ) Number of sorted Sca-1 high+ cells after 11 days of culture with or without BNP or CNP and PKG or p38 inhibitor. n = at least 6 different experiments per group. The data were related to the average of untreated cells. All the results are means ± SEM, *p < 0.05 versus untreated cells and ° p < 0.05 versus BNP or CNP treated cells. ( B ) Summary of the hypothetical signaling pathway induced by BNP to stimulate Sca-1 + cell proliferation. BNP secreted by cardiac cells binds to NPR-B on Sca-1 + cells which increases the cGMP level and activates the protein kinase G (PKG). Furthermore, BNP phosphorylates the p38 MAP kinase. Addition of the SB203580 increases cell proliferation.

Article Snippet: Thus, after isolation, at confluence, the cells were diluted 1:3 before to be either sorted for their Sca-1 expression or directly treated with BNP (1 microM murine BNP (recombinant mouse C 1–45 peptide; American Peptide Co)), CNP (1 microM C-type Natriuretic Peptide (recombinant CNP-22 peptide; Phoenix Pharmaceuticals, Inc)) or ANP (1 microM, Phoenix Pharmaceuticals, Inc).

Techniques:

Journal: The American Journal of Pathology

Article Title: Experimental Autoimmune Breast Failure

doi: 10.1016/j.ajpath.2012.05.025

Figure Lengend Snippet: α-Lactalbumin immunization induces a proinflammatory type-1 immune response. A: Western blot analysis to detect His-tagged mouse α-lactalbumin. Arrows show the positions of the α-lactalbumin monomer and dimer. Positions of molecular weight markers are indicated on the left side of the gel. B: SWXJ female mice were immunized with 150 μg of recombinant mouse α-lactalbumin in CFA. Ten days later, LNCs showed antigen-specific proliferation in recall responses over a broad dose range to α-lactalbumin but not to recombinant human cochlin, an irrelevant control antigen cloned and purified in an identical manner. C: ELISA of culture supernatants from 10-day LNCs stimulated with 20 μg/mL of α-lactalbumin showed production of the type 1 proinflammatory cytokines IFN-γ and IL-2 with minimal production of the type 2 regulatory cytokines IL-5 and IL-10. D: Recall responses to 20 μg/mL of α-lactalbumin were elicited from CD4+ and CD8+ T cells purified by magnetic bead separation from LNCs 10 days after immunization with α-lactalbumin. Data are given as mean ± SE.

Article Snippet: At 8 to 10 weeks of age, mice were injected s.c. in the abdominal flank with 150 μg of recombinant mouse α-lactalbumin in 200 μL of emulsion of equal volumes of water and complete Freund's adjuvant (CFA) containing 400 μg of Mycobacteria tuberculosis H37RA (Difco, Detroit, MI).

Techniques: Western Blot, Molecular Weight, Recombinant, Clone Assay, Purification, Enzyme-linked Immunosorbent Assay

Journal: The American Journal of Pathology

Article Title: Experimental Autoimmune Breast Failure

doi: 10.1016/j.ajpath.2012.05.025

Figure Lengend Snippet: α-Lactalbumin immunization causes infiltration of CD3+ T cells in lactating breast tissue only. Immunohistochemical analysis shows numerous CD3+ T cells clustered in periductal areas (arrows) of breast tissues from lactating mice immunized with α-lactalbumin 6 to 7 weeks before initiation of lactation (top left panel). Clusters of CD3+ T cells were not observed in breast tissues from lactating mice immunized with CFA (top right panel) or in nonlactating mice immunized with either α-lactalbumin (bottom left panel) or CFA (bottom right panel). Scale bars: 50 μm.

Article Snippet: At 8 to 10 weeks of age, mice were injected s.c. in the abdominal flank with 150 μg of recombinant mouse α-lactalbumin in 200 μL of emulsion of equal volumes of water and complete Freund's adjuvant (CFA) containing 400 μg of Mycobacteria tuberculosis H37RA (Difco, Detroit, MI).

Techniques: Immunohistochemical staining

Journal: The American Journal of Pathology

Article Title: Experimental Autoimmune Breast Failure

doi: 10.1016/j.ajpath.2012.05.025

Figure Lengend Snippet: Altered gene expression in lactating breast tissues from α-lactalbumin–immunized mice. Six weeks after immunization with either α-lactalbumin or CFA alone, total RNA from lactating mammary tissue was analyzed for gene expression by real-time qRT-PCR. A: Lactating breast tissue from α-lactalbumin–immunized mice showed significantly elevated gene expression levels of IFN-γ (P = 0.001) but not IL-10 (P > 0.10) and significantly increased gene expression of α-lactalbumin (P < 0.002) and α-casein (P = 0.0008). Asterisks indicate significance. B: α-Lactalbumin and α-casein gene expression levels were not elevated in nonlactating breast tissue from α-lactalbumin–immunized mice. C: Except for the spleen, IFN-γ gene expression levels were not elevated in any tissues examined from normal nonlactating α-lactalbumin–immunized mice. Data are given as mean ± SE.

Article Snippet: At 8 to 10 weeks of age, mice were injected s.c. in the abdominal flank with 150 μg of recombinant mouse α-lactalbumin in 200 μL of emulsion of equal volumes of water and complete Freund's adjuvant (CFA) containing 400 μg of Mycobacteria tuberculosis H37RA (Difco, Detroit, MI).

Techniques: Expressing, Quantitative RT-PCR

Journal: The American Journal of Pathology

Article Title: Experimental Autoimmune Breast Failure

doi: 10.1016/j.ajpath.2012.05.025

Figure Lengend Snippet: Functional phenotype of autoimmune breast failure. Four weeks after immunization with either α-lactalbumin or CFA alone, females were mated with identical males, and the resultant litters were examined daily for differences in mean pup weight. A: Pups from α-lactalbumin–immunized mothers consistently showed a failure to thrive as measured by significantly decreased mean pup weights over several mating cycles (P < 0.04 for every individual mating). Asterisks indicate significance. B: There were no significant differences (P > 0.60) in mean number of mice per litter between females immunized with α-lactalbumin or CFA alone. Nourishment failure often presented with kwashiorkor-like signs, including alopecia (three pups on the left) (C) and liver toxicity as indicated by increased serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) levels (D). Data are given as mean ± SE.

Article Snippet: At 8 to 10 weeks of age, mice were injected s.c. in the abdominal flank with 150 μg of recombinant mouse α-lactalbumin in 200 μL of emulsion of equal volumes of water and complete Freund's adjuvant (CFA) containing 400 μg of Mycobacteria tuberculosis H37RA (Difco, Detroit, MI).

Techniques: Functional Assay

Journal: The American Journal of Pathology

Article Title: Experimental Autoimmune Breast Failure

doi: 10.1016/j.ajpath.2012.05.025

Figure Lengend Snippet: Passive transfer of autoimmune breast failure with primed T cells. Lactating female SWXJ mice were injected i.p. with LNCs, CD4+ T cells, CD8+ T cells, B cells, or sera from mice immunized previously with either recombinant α-lactalbumin or recombinant cochlin in CFA. After weaning (approximately 3 to 4 weeks after transfer), mice were euthanized for molecular analysis of tissues. A: Inhibition of growth did not occur in pups from mothers that received B cells (P > 0.50; left panel) or sera (P > 0.60; middle panel) from α-lactalbumin–immunized mice but did occur in pups from mothers that received α-lactalbumin–activated LNCs (P < 0.04; right panel). B: Significantly elevated α-casein gene expression, a surrogate marker for breast failure, did not occur in lactating breast tissues from control females receiving cochlin-activated LNCs (P > 0.10) but did occur in lactating breast tissues from females receiving α-lactalbumin–activated LNCs (P = 0.02), CD4+ T cells (P = 0.03), or CD8+ T cells (P = 0.02). Data are given as mean ± SE. Asterisks indicate significance. In all experiments, n = 6.

Article Snippet: At 8 to 10 weeks of age, mice were injected s.c. in the abdominal flank with 150 μg of recombinant mouse α-lactalbumin in 200 μL of emulsion of equal volumes of water and complete Freund's adjuvant (CFA) containing 400 μg of Mycobacteria tuberculosis H37RA (Difco, Detroit, MI).

Techniques: Injection, Recombinant, Inhibition, Expressing, Marker

Journal: Cell reports

Article Title: AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences

doi: 10.1016/j.celrep.2023.112903

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal Rabbbit anti-TTR (prealbumin) AbCam Cat # ab75815; RRID:AB_1310604 Monoclonal Rabbit anti-Insulin Cell Signaling Cat # 3014; RRID:AB_2126503 Polyclonal Rabbit anti-mCherry Rockland Cat # 600-401-P16; RRID:AB_2614470 Monoclonal Rabbit anti- p -Akt (Ser473) Cell Signaling Cat # 4060; RRID:AB_2315049 Monoclonal Mouse anti-Alpha1 Na+/K+ ATPase AbCam Cat # ab7671; RRID:AB_306023 Bacterial and virus strains AAV5-CMV-CRE Vector Biolabs Cat # 7012 AAV5-CMV-GFP Vector Biolabs Cat # 7006 AAV5-CMV-Ins2 Vector Biolabs Cat # AAV-262269 AAV5-CMV-GCaMP6f Vector Biolabs Built to order AAV5-TTR-hM3Dq-mCherry Vector Biolabs Built to order AAV5-CMV-Psck1 Vector Biolabs Cat # AAV-268239 AAV5-CMV-Pcsk2 Vector Biolabs Cat # AAV-268242 Chemicals, peptides, and recombinant proteins Compound 21 TOCRIS Cat # 5548 Critical commercial assays PicoPure RNA Isolation Kit ThermoFisher Cat # KIT0204 Ultra Sensitive Insulin ELISA Kit CrystalChem Cat # 90080 Deposited data Raw and analyzed data of RNA-seq This paper GEO: GSE218188 Experimental models: Cell lines iPS(IMR90)-4 WiCell WISCi004-B Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory Strain # 000664 Ai14 The Jackson Laboratory Strain #007914 Ins1 −/− Ins2 fl/fl This paper N/A Software and algorithms ImageJ NIH https://imagej.nih.gov/ij/ GraphPad Prism GraphPad https://graphpad.com R R Foundation for Statistical Computing https://www.r-project.org Other Normal chow diet Dyets Inc Cat# 101845 High fat/high sugar diet Dyets Inc Cat# 103806 Open in a separate window KEY RESOURCES TABLE.

Techniques: Virus, Plasmid Preparation, Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Software

Journal: bioRxiv

Article Title: Heterologous Surface Display Reveals Conserved Complement Inhibition and Functional Diversification of Borrelia burgdorferi Elp Proteins

doi: 10.1101/2024.08.23.609448

Figure Lengend Snippet: (A). Far western blotting was performed as described (Methods), using 2 µg/mL C1s. Binding was detected using mouse anti-C1s antibody and anti-mouse HRP-conjugated antibody. (B.) Strains expressing ElpQ-INPN, ElpQ N-INPN, and ElpQ C-INPN were grown overnight with no induction as previously described. 8×10 6 cells were collected from cultures to add in quadruplicate to C1 and BSA-coated wells (along with PBS). Cells were centrifuged to encourage cell-well contact, and allowed to incubate for 1 hr. Afterwards non-adherent cells were washed away as before, and adherent cells quantified as before. ElpQ and ElpQ mutant binding was assessed in triplicate experiments (n=3). Error bars indicate SEM. ****P < 0.0001; *P < 0.05; ns, not significant using Student’s t test to compare mean values.

Article Snippet: Proteins were incubated overnight with 2 µg/mL of the ligand (in protein-binding buffer), washed, and treated with either mouse anti-FH (Santa Cruz Biotechnology sc-53067) (1:50 dilution in Bio-Rad EveryBlot) or mouse anti-C1s (R&D Systems MAB2060), followed by anti-mouse HRP antibody (Promega W4021).

Techniques: Far Western Blot, Binding Assay, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Heterologous Surface Display Reveals Conserved Complement Inhibition and Functional Diversification of Borrelia burgdorferi Elp Proteins

doi: 10.1101/2024.08.23.609448

Figure Lengend Snippet: (A.) Percentage of FITC positivity for various strains grown under the induction condition and analyzed through flow cytometry using rabbit anti-His and anti-rabbit AF488 antibodies. Positivity determined as previously described. (B). Strains expressing ElpQ-INPN and the four selected Elp homologs linked to INP were grown to ∼0.6 OD 600 and induced for 18 hr as previously described. 8×10 8 cells were collected to add in quadruplicate to C1s and BSA-coated wells. Whole cell ELISA was performed as previously described in a single experiment (n=1). Error bars indicate SEM. ****P < 0.0001; *P < 0.05; ns, not significant using Student’s t test to compare mean values. (C). ElpB and Elp homolog recombinant proteins, along with GST, were added in a dilution series starting at a concentration of 2 μM to wells coated with C1s and BSA. ELISA was carried out as previously described with protein binding quantified through anti-GST and anti-GST HRP incubation. Each recombinant protein was assessed at least in triplicate (n=3). The highest BSA background mean value assessed was 0.256 OD 450 . Error bars indicate SEM. ****P < 0.0001; *P < 0.05; ns, not significant using Student’s t test to compare mean values. (D). Each Elp homolog was immobilized on an SPR sensor chip. Purified activated human C1s was injected over each biosensor in a two-fold dilution series ranging from 0 to 200 nM. Sensorgrams from a representative injection series are shown. Each injection series was performed in triplicate (n=3). Sensorgrams were fit to a 1:1 kinetic model of binding (Langmuir). The raw sensorgrams are shown in black and the kinetic fits in red.

Article Snippet: Proteins were incubated overnight with 2 µg/mL of the ligand (in protein-binding buffer), washed, and treated with either mouse anti-FH (Santa Cruz Biotechnology sc-53067) (1:50 dilution in Bio-Rad EveryBlot) or mouse anti-C1s (R&D Systems MAB2060), followed by anti-mouse HRP antibody (Promega W4021).

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Protein Binding, Incubation, Purification, Injection, Binding Assay

Journal: bioRxiv

Article Title: Heterologous Surface Display Reveals Conserved Complement Inhibition and Functional Diversification of Borrelia burgdorferi Elp Proteins

doi: 10.1101/2024.08.23.609448

Figure Lengend Snippet: (A). Strains expressing ElpQ- INPN and the four selected Elp homologs linked to INP were grown to ∼0.6 OD 600 and induced for 18 hr as previously described. 8×10 8 cells were collected to add in quadruplicate to C1r and BSA-coated wells. Whole cell ELISA was performed as previously described in a single experiment (n=1). Error bars indicate SEM. ****P < 0.0001; *P < 0.05; ns, not significant using Student’s t test to compare mean values. (B). BBK32 and Elp homolog recombinant proteins, along with GST, were added in a dilution series starting at a concentration of 2 μM to wells coated with C1r and BSA. ELISA was carried out as previously described with protein binding quantified through anti-GST and anti-GST HRP incubation. BG032 and BG038 recombinant proteins were assessed at least in triplicate (n=3). BG036 and BG044 recombinant proteins were assessed with one replicate (n=1). Error bars indicate SEM. ****P < 0.0001; *P < 0.05; ns, not significant using Student’s t test to compare mean values. The highest BSA background mean value assessed was 0.256 OD 450 . (C). Each Elp homolog was immobilized on an SPR sensor chip. Purified activated human C1s was injected over each biosensor in a two-fold dilution series ranging from 0 to 200 nM. Sensorgrams from a representative injection series are shown. Each injection series was performed in triplicate (n=3).

Article Snippet: Proteins were incubated overnight with 2 µg/mL of the ligand (in protein-binding buffer), washed, and treated with either mouse anti-FH (Santa Cruz Biotechnology sc-53067) (1:50 dilution in Bio-Rad EveryBlot) or mouse anti-C1s (R&D Systems MAB2060), followed by anti-mouse HRP antibody (Promega W4021).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Protein Binding, Incubation, Purification, Injection