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R&D Systems human c1r
Bacterial Strains and Plasmid Constructs used in this study.

Human C1r, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human c1r - by Bioz Stars, 2026-07

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1) Product Images from "Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions"

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.886733

Figure Legend Snippet: Bacterial Strains and Plasmid Constructs used in this study.

Techniques Used: Plasmid Preparation, Construct, Control, Knock-In, Over Expression, Recombinant

B. miyamotoi encodes two orthologous genes to B. burgdorferi BBK32. (A) BBK32 orthologs are found in relapsing fever (RF)-associated and B. miyamotoi spirochetes and are denoted FbpA, FbpB, and FbpC . B. miyamotoi FR64b FbpA and FbpB are underlined. (B) An alignment of B. miyamotoi strain FR64b FbpA and FbpB to B. burgdorferi strain B31 BBK32 shows differences of the amino acid sequences within the fibronectin binding (green box) and complement inhibitory domains (blue box). The gelatin-binding domain (GBD) of BBK32 is denoted. The key residues R248 and K327 of BBK32 involved in complement C1r binding are indicated by a red box. * conserved, : strongly similar, . weakly similar.

Figure Legend Snippet: B. miyamotoi encodes two orthologous genes to B. burgdorferi BBK32. (A) BBK32 orthologs are found in relapsing fever (RF)-associated and B. miyamotoi spirochetes and are denoted FbpA, FbpB, and FbpC . B. miyamotoi FR64b FbpA and FbpB are underlined. (B) An alignment of B. miyamotoi strain FR64b FbpA and FbpB to B. burgdorferi strain B31 BBK32 shows differences of the amino acid sequences within the fibronectin binding (green box) and complement inhibitory domains (blue box). The gelatin-binding domain (GBD) of BBK32 is denoted. The key residues R248 and K327 of BBK32 involved in complement C1r binding are indicated by a red box. * conserved, : strongly similar, . weakly similar.

Techniques Used: Binding Assay

Assessing the interaction of human C1r with B. miyamotoi FbpA and FbpB. SPR was used to assess protein-protein interactions between the C-terminal regions of each Fbp protein and activated C1r-CCP2-SP. Immobilized (A) FbpA-C, (B) FbpB-C, and (C) FbpA-C-R264A-K343A (referred to as FbpA DA-C throughout) were subjected to an injection series of serially diluted C1r-CCP2-SP (0.78 - 100 nM). A representative sensorgram from the three replicates for each Fbp is shown with the black curve being the sensorgram and the red curve the associated kinetic fit. K D values were determined using kinetic fits and are shown as the mean +/- standard deviation of three replicates in <xref ref-type=

Table 4 . A K D value for C1r-CCP2-SP interactions with FbpA DA-C was not determined (N.D.). (D, E) Competition experiments were carried out using 25 nM C1r-CCP2-SP alone or mixed with 25 nM soluble Fbp proteins injected over FbpA-C (D) or FbpB-C (E) . (F) Comparisons of statistical significance were performed for data shown in panels (D, E) using a one-way ANOVA followed by a multiple comparison Tukey test (* = p < 0.05). " title="Assessing the interaction of human C1r with B. miyamotoi FbpA and FbpB. SPR was ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Assessing the interaction of human C1r with B. miyamotoi FbpA and FbpB. SPR was used to assess protein-protein interactions between the C-terminal regions of each Fbp protein and activated C1r-CCP2-SP. Immobilized (A) FbpA-C, (B) FbpB-C, and (C) FbpA-C-R264A-K343A (referred to as FbpA DA-C throughout) were subjected to an injection series of serially diluted C1r-CCP2-SP (0.78 - 100 nM). A representative sensorgram from the three replicates for each Fbp is shown with the black curve being the sensorgram and the red curve the associated kinetic fit. K D values were determined using kinetic fits and are shown as the mean +/- standard deviation of three replicates in Table 4 . A K D value for C1r-CCP2-SP interactions with FbpA DA-C was not determined (N.D.). (D, E) Competition experiments were carried out using 25 nM C1r-CCP2-SP alone or mixed with 25 nM soluble Fbp proteins injected over FbpA-C (D) or FbpB-C (E) . (F) Comparisons of statistical significance were performed for data shown in panels (D, E) using a one-way ANOVA followed by a multiple comparison Tukey test (* = p < 0.05).

Techniques Used: Protein-Protein interactions, Injection, Standard Deviation, Comparison

Figure Legend Snippet: SPR and complement assay results.

Techniques Used: Complement Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

FbpA-C and FbpB-C interact differentially with zymogen and activated forms of human C1r. (A) Single cycle SPR was used to determine binding affinities of C1r zymogen or active C1r ranging from 0.16 - 100 nM injected over immobilized Fbps. A representative sensorgram from a three-injection series for each Fbp-C and C1r active state is shown with the black curve being the sensorgram and the red curve the associated kinetic fit. K D ’s were determined using kinetic fits and are shown as the average and standard deviation of three replicates in <xref ref-type=

Table 4 . (B) Far Western overlays using B. burgdorferi B314 lysates probed with zymogen or enzymatic forms of purified full-length C1r, followed by an antibody to C1r. (C) An ELISA-type binding assay was used to determine the ability of FbpA-C, FbpA DA-C and FbpB-C to interact with C1r in human serum. FbpA-C (black), FbpB-C (blue), and FbpA DA-C (red) were immobilized on an ELISA plate then incubated with a two-fold dilution series of normal human serum ranging from 0.0012-10%. Corresponding EC 50 values for FbpA-C, FbpB-C, and FbpA DA-C are reported in Table 4 . (D) Fbp-mediated inhibition of purified active C1r-CCP2-SP enzymatic activity was assessed by incubating 15 nM C1r-CCP2-SP with a 1 μM of FbpA-C (white), FbpB-C (striped), and FbpA DA-C (grey). C1r-CCP2-SP enzymatic activity was determined by incubation with a C1r substrate (Z-Gly-Arg-sBzl) that, once cleaved, reacts with DTNB resulting in a colorimetric change. No statistical difference was found between FbpA-C and FbpB-C inhibitory activity, whereas FbpA DA-C exhibited significant loss in inhibitory activity. Statistical analysis was performed using a one-way ANOVA followed by a multiple comparison Tukey test (*=p<0.05). " title="... differentially with zymogen and activated forms of human C1r. (A) Single cycle SPR was used to determine ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: FbpA-C and FbpB-C interact differentially with zymogen and activated forms of human C1r. (A) Single cycle SPR was used to determine binding affinities of C1r zymogen or active C1r ranging from 0.16 - 100 nM injected over immobilized Fbps. A representative sensorgram from a three-injection series for each Fbp-C and C1r active state is shown with the black curve being the sensorgram and the red curve the associated kinetic fit. K D ’s were determined using kinetic fits and are shown as the average and standard deviation of three replicates in Table 4 . (B) Far Western overlays using B. burgdorferi B314 lysates probed with zymogen or enzymatic forms of purified full-length C1r, followed by an antibody to C1r. (C) An ELISA-type binding assay was used to determine the ability of FbpA-C, FbpA DA-C and FbpB-C to interact with C1r in human serum. FbpA-C (black), FbpB-C (blue), and FbpA DA-C (red) were immobilized on an ELISA plate then incubated with a two-fold dilution series of normal human serum ranging from 0.0012-10%. Corresponding EC 50 values for FbpA-C, FbpB-C, and FbpA DA-C are reported in Table 4 . (D) Fbp-mediated inhibition of purified active C1r-CCP2-SP enzymatic activity was assessed by incubating 15 nM C1r-CCP2-SP with a 1 μM of FbpA-C (white), FbpB-C (striped), and FbpA DA-C (grey). C1r-CCP2-SP enzymatic activity was determined by incubation with a C1r substrate (Z-Gly-Arg-sBzl) that, once cleaved, reacts with DTNB resulting in a colorimetric change. No statistical difference was found between FbpA-C and FbpB-C inhibitory activity, whereas FbpA DA-C exhibited significant loss in inhibitory activity. Statistical analysis was performed using a one-way ANOVA followed by a multiple comparison Tukey test (*=p<0.05).

Techniques Used: Binding Assay, Injection, Standard Deviation, Western Blot, Purification, Enzyme-linked Immunosorbent Assay, Incubation, Inhibition, Activity Assay, Comparison

Determining the ability of surface-expressed B. miyamotoi FbpA and FbpB to protect a serum-sensitive strain of B. burgdorferi . (A) Western blots of lysates from B. miyamotoi strain FR64b and B burgdorferi B314 isolates expressing fbpA , fbpA-DA , fbpB , and bbk32 were probed with antibodies to FbpA (anti-FbpA), FbpB (anti-FbpB), BBK32 (anti-BBK32) and FlaB (anti-FlaB), the latter as a loading control and a control for a subsurface target. Vector refers to the plasmid-only backbone sample (B314/pBBE22 luc ). Samples from each population were subjected to proteinase K accessibility treatments to determine the surface expression of the proteins. Due to its periplasmic location, the flagellar protein, FlaB, is unaffected by proteinase K in intact cells and depicts structural integrity in the treated B. miyamotoi and B314 derivatives listed. (B) B. miyamotoi Fbp proteins were tested for their ability to confer resistance to normal human serum (NHS) sensitive B. burgdorferi strain B314 relative to vector-only and BBK32-expressing controls (negative and positive controls, respectively). Asterisks depict a significant increase in survival relative to FbpA DA and the vector control (*=p<0.0001). All strains exposed to heat inactivated NHS (hiNHS), rendering complement proteins inactive, were largely unaffected. (C) Rescue assays were used to determine if soluble exogenous recombinant proteins could promote survival of serum-sensitive vector-containing B. burgdorferi B314. Increasing five-fold concentrations of BBK32-C, FbpA-C, FbpA DA-C, and FbpB-C were added in serum sensitivity assays. The middle samples (i.e., 240 nM) have added recombinant protein that is roughly equivalent to the concentration of C1r in the assay conditions employed. Cells were assessed via darkfield microscopy. Included as controls are a vector-only strain of B314 with no exogenous protein addition, as well as a BBK32-producing B314 isolate (right side). Asterisks depict a significant increase in survival relative to the vector control (*=p<0.015). Significance for serum sensitivity and rescue assays was determined using two-way ANOVA with a Šidák correction for multiple comparisons.

Figure Legend Snippet: Determining the ability of surface-expressed B. miyamotoi FbpA and FbpB to protect a serum-sensitive strain of B. burgdorferi . (A) Western blots of lysates from B. miyamotoi strain FR64b and B burgdorferi B314 isolates expressing fbpA , fbpA-DA , fbpB , and bbk32 were probed with antibodies to FbpA (anti-FbpA), FbpB (anti-FbpB), BBK32 (anti-BBK32) and FlaB (anti-FlaB), the latter as a loading control and a control for a subsurface target. Vector refers to the plasmid-only backbone sample (B314/pBBE22 luc ). Samples from each population were subjected to proteinase K accessibility treatments to determine the surface expression of the proteins. Due to its periplasmic location, the flagellar protein, FlaB, is unaffected by proteinase K in intact cells and depicts structural integrity in the treated B. miyamotoi and B314 derivatives listed. (B) B. miyamotoi Fbp proteins were tested for their ability to confer resistance to normal human serum (NHS) sensitive B. burgdorferi strain B314 relative to vector-only and BBK32-expressing controls (negative and positive controls, respectively). Asterisks depict a significant increase in survival relative to FbpA DA and the vector control (*=p<0.0001). All strains exposed to heat inactivated NHS (hiNHS), rendering complement proteins inactive, were largely unaffected. (C) Rescue assays were used to determine if soluble exogenous recombinant proteins could promote survival of serum-sensitive vector-containing B. burgdorferi B314. Increasing five-fold concentrations of BBK32-C, FbpA-C, FbpA DA-C, and FbpB-C were added in serum sensitivity assays. The middle samples (i.e., 240 nM) have added recombinant protein that is roughly equivalent to the concentration of C1r in the assay conditions employed. Cells were assessed via darkfield microscopy. Included as controls are a vector-only strain of B314 with no exogenous protein addition, as well as a BBK32-producing B314 isolate (right side). Asterisks depict a significant increase in survival relative to the vector control (*=p<0.015). Significance for serum sensitivity and rescue assays was determined using two-way ANOVA with a Šidák correction for multiple comparisons.

Techniques Used: Western Blot, Expressing, Control, Plasmid Preparation, Recombinant, Concentration Assay, Microscopy

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Figure 2. Characterization of the anti-NKp46 mAb 09. (A) FACS staining of anti-NKp46 mAbs (9E2 and 09) binding to BW parental cells versus BW transfected cells expressing NKp46 (black and red histograms, respectively). Binding was detected using an anti-mouse IgG AF647 labeled antibody. The ﬁlled gray histogram represents staining with secondary antibodies only of the BW parental cells. The background (BG) of BW NKp46 transfectants was similar and is not shown in the ﬁgure. The ﬁgure shows one representative experiment out of the six performed. (B) FACS staining of anti-NKp46 mAbs (9E2 and 09) on primary activated bulk human NK cells (black histogram). The ﬁlled gray histogram represents the staining of NK cells with secondary antibodies only. The ﬁgure shows one representative experiment out of the six performed. (C) Human NKp46-Ig was pre-incubated either alone (black histogram) or with anti-NKp46 mAbs (9E2 and 09, red histograms) at 4 ◦C, followed by FACS staining of BJAB, MCF7, and C1R cells detected with an anti-human IgG PE-labeled antibody. The ﬁlled gray histogram represents the staining of cells with secondary antibodies only. The ﬁgure shows one representative experiment out of the two performed. (D) Activated bulk NK cell cultures were incubated with the indicated anti-NKp46 mAbs (9E2 and 09) at 4 ◦C (black histogram) or 37 ◦C (red histogram) for 8 h, followed by FACS staining with an AF647-labeled anti-mouse secondary antibody. The ﬁlled gray histogram represents staining with secondary antibodies only of cells treated at 4 ◦C. The background of cells treated at 37 ◦C was similar and is not shown in the ﬁgure. The ﬁgure shows one representative experiment out of the ﬁve performed.

Journal: Cells

Article Title: Derivation and Preclinical Characterization of CYT-303, a Novel NKp46-NK Cell Engager Targeting GPC3.

doi: 10.3390/cells12070996

Figure Lengend Snippet: Figure 2. Characterization of the anti-NKp46 mAb 09. (A) FACS staining of anti-NKp46 mAbs (9E2 and 09) binding to BW parental cells versus BW transfected cells expressing NKp46 (black and red histograms, respectively). Binding was detected using an anti-mouse IgG AF647 labeled antibody. The ﬁlled gray histogram represents staining with secondary antibodies only of the BW parental cells. The background (BG) of BW NKp46 transfectants was similar and is not shown in the ﬁgure. The ﬁgure shows one representative experiment out of the six performed. (B) FACS staining of anti-NKp46 mAbs (9E2 and 09) on primary activated bulk human NK cells (black histogram). The ﬁlled gray histogram represents the staining of NK cells with secondary antibodies only. The ﬁgure shows one representative experiment out of the six performed. (C) Human NKp46-Ig was pre-incubated either alone (black histogram) or with anti-NKp46 mAbs (9E2 and 09, red histograms) at 4 ◦C, followed by FACS staining of BJAB, MCF7, and C1R cells detected with an anti-human IgG PE-labeled antibody. The ﬁlled gray histogram represents the staining of cells with secondary antibodies only. The ﬁgure shows one representative experiment out of the two performed. (D) Activated bulk NK cell cultures were incubated with the indicated anti-NKp46 mAbs (9E2 and 09) at 4 ◦C (black histogram) or 37 ◦C (red histogram) for 8 h, followed by FACS staining with an AF647-labeled anti-mouse secondary antibody. The ﬁlled gray histogram represents staining with secondary antibodies only of cells treated at 4 ◦C. The background of cells treated at 37 ◦C was similar and is not shown in the ﬁgure. The ﬁgure shows one representative experiment out of the ﬁve performed.

Article Snippet: Hep3B (human hepatocarcinoma), HepG2 (human hepatoblastoma), Huh-7 (human hepatocarcinoma), BW (murine T-lymphoblast), BJAB (EBV-negative human Burkitt-like lymphoma), MCF7 (human breast cancer), and C1R (human B-cell lymphoblastoid) cell lines used in this study were purchased from ATCC (Manassas, VA, USA).

Techniques: Staining, Binding Assay, Transfection, Expressing, Labeling, Incubation

Figure 2. Identiﬁcation of TCRs of FGFR3Y373C-speciﬁc CD8+ T cells in a large-scale screening. (A) A large-scale screening of mutant FGFR3Y373C peptide-speciﬁc CD8+ T cells by IFN-γ ELISPOT assay. FGFR3Y373C antigen-stimulated CD8+ T cells were co-cultured with C1R-A0206 cells pulsed with FGFR3Y373C or wild-type FGFR3WT peptide. (B) IFN-γ secretion of expanded FGFR3Y373C-specific

Journal: Cancers

Article Title: Identification of T Cell Receptors Targeting a Neoantigen Derived from Recurrently Mutated FGFR3.

doi: 10.3390/cancers15041031

Figure Lengend Snippet: Figure 2. Identiﬁcation of TCRs of FGFR3Y373C-speciﬁc CD8+ T cells in a large-scale screening. (A) A large-scale screening of mutant FGFR3Y373C peptide-speciﬁc CD8+ T cells by IFN-γ ELISPOT assay. FGFR3Y373C antigen-stimulated CD8+ T cells were co-cultured with C1R-A0206 cells pulsed with FGFR3Y373C or wild-type FGFR3WT peptide. (B) IFN-γ secretion of expanded FGFR3Y373C-specific

Article Snippet: We purchased C1R (B lymphoblasts lacking endogenous human leukocyte antigen (HLA)-A and HLA-B expression), Jiyoye, and EB-3 cells from the American Type Culture Collection (Rockville, MD, USA) and cultured them in RPMI1640 supplemented with 10% FBS.

Techniques: Mutagenesis, Enzyme-linked Immunospot, Cell Culture

Immunohistochemical analysis of C4 convertases of the classical complement pathway in placental tissue. Sections of pre-eclamptic (PE) and normal (CONTROL) placentae were stained for C1r and C1s. The panel shows representative images of PE and control placentae documenting complete absence of these C components in both groups of placentae. Scale bars, 50 μm.

Journal: Frontiers in Immunology

Article Title: Distinct Roles of Classical and Lectin Pathways of Complement in Preeclamptic Placentae

doi: 10.3389/fimmu.2022.882298

Figure Lengend Snippet: Immunohistochemical analysis of C4 convertases of the classical complement pathway in placental tissue. Sections of pre-eclamptic (PE) and normal (CONTROL) placentae were stained for C1r and C1s. The panel shows representative images of PE and control placentae documenting complete absence of these C components in both groups of placentae. Scale bars, 50 μm.

Article Snippet: C1r , human , 1:100 , rabbit pAb , HPA001551 , Sigma Aldrich.

Techniques: Immunohistochemical staining, Control, Staining

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: Bacterial Strains and Plasmid Constructs used in this study.

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Plasmid Preparation, Construct, Control, Knock-In, Over Expression, Recombinant

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: B. miyamotoi encodes two orthologous genes to B. burgdorferi BBK32. (A) BBK32 orthologs are found in relapsing fever (RF)-associated and B. miyamotoi spirochetes and are denoted FbpA, FbpB, and FbpC . B. miyamotoi FR64b FbpA and FbpB are underlined. (B) An alignment of B. miyamotoi strain FR64b FbpA and FbpB to B. burgdorferi strain B31 BBK32 shows differences of the amino acid sequences within the fibronectin binding (green box) and complement inhibitory domains (blue box). The gelatin-binding domain (GBD) of BBK32 is denoted. The key residues R248 and K327 of BBK32 involved in complement C1r binding are indicated by a red box. * conserved, : strongly similar, . weakly similar.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: Assessing the interaction of human C1r with B. miyamotoi FbpA and FbpB. SPR was used to assess protein-protein interactions between the C-terminal regions of each Fbp protein and activated C1r-CCP2-SP. Immobilized (A) FbpA-C, (B) FbpB-C, and (C) FbpA-C-R264A-K343A (referred to as FbpA DA-C throughout) were subjected to an injection series of serially diluted C1r-CCP2-SP (0.78 - 100 nM). A representative sensorgram from the three replicates for each Fbp is shown with the black curve being the sensorgram and the red curve the associated kinetic fit. K D values were determined using kinetic fits and are shown as the mean +/- standard deviation of three replicates in Table 4 . A K D value for C1r-CCP2-SP interactions with FbpA DA-C was not determined (N.D.). (D, E) Competition experiments were carried out using 25 nM C1r-CCP2-SP alone or mixed with 25 nM soluble Fbp proteins injected over FbpA-C (D) or FbpB-C (E) . (F) Comparisons of statistical significance were performed for data shown in panels (D, E) using a one-way ANOVA followed by a multiple comparison Tukey test (* = p < 0.05).

Techniques: Protein-Protein interactions, Injection, Standard Deviation, Comparison

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: SPR and complement assay results.

Techniques: Complement Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: FbpA-C and FbpB-C interact differentially with zymogen and activated forms of human C1r. (A) Single cycle SPR was used to determine binding affinities of C1r zymogen or active C1r ranging from 0.16 - 100 nM injected over immobilized Fbps. A representative sensorgram from a three-injection series for each Fbp-C and C1r active state is shown with the black curve being the sensorgram and the red curve the associated kinetic fit. K D ’s were determined using kinetic fits and are shown as the average and standard deviation of three replicates in Table 4 . (B) Far Western overlays using B. burgdorferi B314 lysates probed with zymogen or enzymatic forms of purified full-length C1r, followed by an antibody to C1r. (C) An ELISA-type binding assay was used to determine the ability of FbpA-C, FbpA DA-C and FbpB-C to interact with C1r in human serum. FbpA-C (black), FbpB-C (blue), and FbpA DA-C (red) were immobilized on an ELISA plate then incubated with a two-fold dilution series of normal human serum ranging from 0.0012-10%. Corresponding EC 50 values for FbpA-C, FbpB-C, and FbpA DA-C are reported in Table 4 . (D) Fbp-mediated inhibition of purified active C1r-CCP2-SP enzymatic activity was assessed by incubating 15 nM C1r-CCP2-SP with a 1 μM of FbpA-C (white), FbpB-C (striped), and FbpA DA-C (grey). C1r-CCP2-SP enzymatic activity was determined by incubation with a C1r substrate (Z-Gly-Arg-sBzl) that, once cleaved, reacts with DTNB resulting in a colorimetric change. No statistical difference was found between FbpA-C and FbpB-C inhibitory activity, whereas FbpA DA-C exhibited significant loss in inhibitory activity. Statistical analysis was performed using a one-way ANOVA followed by a multiple comparison Tukey test (*=p<0.05).

Techniques: Binding Assay, Injection, Standard Deviation, Western Blot, Purification, Enzyme-linked Immunosorbent Assay, Incubation, Inhibition, Activity Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: Determining the ability of surface-expressed B. miyamotoi FbpA and FbpB to protect a serum-sensitive strain of B. burgdorferi . (A) Western blots of lysates from B. miyamotoi strain FR64b and B burgdorferi B314 isolates expressing fbpA , fbpA-DA , fbpB , and bbk32 were probed with antibodies to FbpA (anti-FbpA), FbpB (anti-FbpB), BBK32 (anti-BBK32) and FlaB (anti-FlaB), the latter as a loading control and a control for a subsurface target. Vector refers to the plasmid-only backbone sample (B314/pBBE22 luc ). Samples from each population were subjected to proteinase K accessibility treatments to determine the surface expression of the proteins. Due to its periplasmic location, the flagellar protein, FlaB, is unaffected by proteinase K in intact cells and depicts structural integrity in the treated B. miyamotoi and B314 derivatives listed. (B) B. miyamotoi Fbp proteins were tested for their ability to confer resistance to normal human serum (NHS) sensitive B. burgdorferi strain B314 relative to vector-only and BBK32-expressing controls (negative and positive controls, respectively). Asterisks depict a significant increase in survival relative to FbpA DA and the vector control (*=p<0.0001). All strains exposed to heat inactivated NHS (hiNHS), rendering complement proteins inactive, were largely unaffected. (C) Rescue assays were used to determine if soluble exogenous recombinant proteins could promote survival of serum-sensitive vector-containing B. burgdorferi B314. Increasing five-fold concentrations of BBK32-C, FbpA-C, FbpA DA-C, and FbpB-C were added in serum sensitivity assays. The middle samples (i.e., 240 nM) have added recombinant protein that is roughly equivalent to the concentration of C1r in the assay conditions employed. Cells were assessed via darkfield microscopy. Included as controls are a vector-only strain of B314 with no exogenous protein addition, as well as a BBK32-producing B314 isolate (right side). Asterisks depict a significant increase in survival relative to the vector control (*=p<0.015). Significance for serum sensitivity and rescue assays was determined using two-way ANOVA with a Šidák correction for multiple comparisons.

Techniques: Western Blot, Expressing, Control, Plasmid Preparation, Recombinant, Concentration Assay, Microscopy

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