anti cbd monoclonal antibody  (New England Biolabs)


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    New England Biolabs anti cbd monoclonal antibody
    Effects of vIRF-1 on complexing of ISGF3 components. (A-C) Flag-tagged STAT1 (A) or IRF9 (C) or <t>CBD-tagged</t> STAT2 (B) were expressed in 293T cells transfected with the respective expression plasmids and either vIRF-1 (virf1) or empty (-) expression vector; replicates were either left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h. Flag/CBD-tagged proteins were then immuno/affinity-precipitated from cell lysates, and coprecipitated ISGF3 proteins were detected by immunoblotting. Diagrams below the panels illustrate the main findings from each experiment. (D-E) Serial coprecipitations of STAT1 and STAT2 (D), STAT1 and IRF9 (E), and IRF9 and STAT2 (F), respectively Flag- and CBD-tagged, from lysates of transfected 293T cells expressing vIRF-1 (virf1) or cotransfected with empty vector (-). All transfectants were treated with IFNβ for 24 h prior to harvesting. First (Flag IP) and second (CBD AP) precipitates (Precip. 1, Precip. 2) were analyzed by immunoblotting for the tagged “bait” proteins, the third (endogenous) ISGF3 protein (including phosphorylated and total STAT1 and STAT2), and vIRF-1; lysates were immunoblotted for expression of input proteins. Illustrated below each panel of blots are the main findings. (G) Disruption by vIRF-1 of STAT1-STAT2 complexes isolated by Flag-IP (STAT1) and CBD-AP (STAT2) from IFNβ-treated transfected 293T cells. Immunoprecipitated material from vIRF-1-Flag (virf1) or empty control (cntl) vector-transfected cells was applied in two concentrations (1x, 2x) to dual-precipitation-derived STAT1/STAT2 complexes, and then mixtures were subjected to re-precipitation with chitin beads (binding STAT2-CBD). STAT1 and vIRF-1 associated with re-precipitated STAT2-CBD were identified by immunoblotting. Relative levels of co-precipitated STAT1, normalized to affinity-sedimented STAT2, are shown below the STAT1 blot (cntl/1x value set at 1). (H) An equivalent experiment was carried out using GST-fused recombinant vIRF-1 (virf1) or GST (negative control) to challenge STAT1 interaction with STAT2 in STAT1/STAT2 hetero-complexes isolated by IP/AP dual precipitations from IFNβ-treated 293T cells. Endogenous IRF9 interaction with STAT1/2 and competition by vIRF-1 were also monitored. Relative levels and integrities of the recombinant proteins are shown in the Coomassie-stained gel (right); arrowheads indicate the positions of the full-length proteins.
    Anti Cbd Monoclonal Antibody, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cbd monoclonal antibody/product/New England Biolabs
    Average 92 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    anti cbd monoclonal antibody - by Bioz Stars, 2022-09
    92/100 stars

    Images

    1) Product Images from "STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling"

    Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1010676

    Effects of vIRF-1 on complexing of ISGF3 components. (A-C) Flag-tagged STAT1 (A) or IRF9 (C) or CBD-tagged STAT2 (B) were expressed in 293T cells transfected with the respective expression plasmids and either vIRF-1 (virf1) or empty (-) expression vector; replicates were either left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h. Flag/CBD-tagged proteins were then immuno/affinity-precipitated from cell lysates, and coprecipitated ISGF3 proteins were detected by immunoblotting. Diagrams below the panels illustrate the main findings from each experiment. (D-E) Serial coprecipitations of STAT1 and STAT2 (D), STAT1 and IRF9 (E), and IRF9 and STAT2 (F), respectively Flag- and CBD-tagged, from lysates of transfected 293T cells expressing vIRF-1 (virf1) or cotransfected with empty vector (-). All transfectants were treated with IFNβ for 24 h prior to harvesting. First (Flag IP) and second (CBD AP) precipitates (Precip. 1, Precip. 2) were analyzed by immunoblotting for the tagged “bait” proteins, the third (endogenous) ISGF3 protein (including phosphorylated and total STAT1 and STAT2), and vIRF-1; lysates were immunoblotted for expression of input proteins. Illustrated below each panel of blots are the main findings. (G) Disruption by vIRF-1 of STAT1-STAT2 complexes isolated by Flag-IP (STAT1) and CBD-AP (STAT2) from IFNβ-treated transfected 293T cells. Immunoprecipitated material from vIRF-1-Flag (virf1) or empty control (cntl) vector-transfected cells was applied in two concentrations (1x, 2x) to dual-precipitation-derived STAT1/STAT2 complexes, and then mixtures were subjected to re-precipitation with chitin beads (binding STAT2-CBD). STAT1 and vIRF-1 associated with re-precipitated STAT2-CBD were identified by immunoblotting. Relative levels of co-precipitated STAT1, normalized to affinity-sedimented STAT2, are shown below the STAT1 blot (cntl/1x value set at 1). (H) An equivalent experiment was carried out using GST-fused recombinant vIRF-1 (virf1) or GST (negative control) to challenge STAT1 interaction with STAT2 in STAT1/STAT2 hetero-complexes isolated by IP/AP dual precipitations from IFNβ-treated 293T cells. Endogenous IRF9 interaction with STAT1/2 and competition by vIRF-1 were also monitored. Relative levels and integrities of the recombinant proteins are shown in the Coomassie-stained gel (right); arrowheads indicate the positions of the full-length proteins.
    Figure Legend Snippet: Effects of vIRF-1 on complexing of ISGF3 components. (A-C) Flag-tagged STAT1 (A) or IRF9 (C) or CBD-tagged STAT2 (B) were expressed in 293T cells transfected with the respective expression plasmids and either vIRF-1 (virf1) or empty (-) expression vector; replicates were either left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h. Flag/CBD-tagged proteins were then immuno/affinity-precipitated from cell lysates, and coprecipitated ISGF3 proteins were detected by immunoblotting. Diagrams below the panels illustrate the main findings from each experiment. (D-E) Serial coprecipitations of STAT1 and STAT2 (D), STAT1 and IRF9 (E), and IRF9 and STAT2 (F), respectively Flag- and CBD-tagged, from lysates of transfected 293T cells expressing vIRF-1 (virf1) or cotransfected with empty vector (-). All transfectants were treated with IFNβ for 24 h prior to harvesting. First (Flag IP) and second (CBD AP) precipitates (Precip. 1, Precip. 2) were analyzed by immunoblotting for the tagged “bait” proteins, the third (endogenous) ISGF3 protein (including phosphorylated and total STAT1 and STAT2), and vIRF-1; lysates were immunoblotted for expression of input proteins. Illustrated below each panel of blots are the main findings. (G) Disruption by vIRF-1 of STAT1-STAT2 complexes isolated by Flag-IP (STAT1) and CBD-AP (STAT2) from IFNβ-treated transfected 293T cells. Immunoprecipitated material from vIRF-1-Flag (virf1) or empty control (cntl) vector-transfected cells was applied in two concentrations (1x, 2x) to dual-precipitation-derived STAT1/STAT2 complexes, and then mixtures were subjected to re-precipitation with chitin beads (binding STAT2-CBD). STAT1 and vIRF-1 associated with re-precipitated STAT2-CBD were identified by immunoblotting. Relative levels of co-precipitated STAT1, normalized to affinity-sedimented STAT2, are shown below the STAT1 blot (cntl/1x value set at 1). (H) An equivalent experiment was carried out using GST-fused recombinant vIRF-1 (virf1) or GST (negative control) to challenge STAT1 interaction with STAT2 in STAT1/STAT2 hetero-complexes isolated by IP/AP dual precipitations from IFNβ-treated 293T cells. Endogenous IRF9 interaction with STAT1/2 and competition by vIRF-1 were also monitored. Relative levels and integrities of the recombinant proteins are shown in the Coomassie-stained gel (right); arrowheads indicate the positions of the full-length proteins.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Isolation, Immunoprecipitation, Derivative Assay, Binding Assay, Recombinant, Negative Control, Staining

    Physical and functional interactions of vIRF-1 with JAKs. (A) TYK2-S and STAT2-CBD were expressed with (virf1) or without (-) vIRF-1 in transfected 293T cells. Cell lysates and S-protein affinity-precipitates were assessed for input protein expression and sedimentation by immunoblotting with CBD (STAT2), vIRF-1, and S-tag (TYK2) antibodies. Affinity-precipitated TYK-2-S was probed with phospho-tyrosine (PY)-specific antibody to identify the active, autophosphorylated form of the kinase. Dotted lines indicate lane deletions from single membranes; the arrowhead and asterisk indicate CBD-specific (STAT2) band and remnant S-tag signal (after blot stripping), respectively. (B) An equivalent experiment was performed to assess vIRF-1 effects on JAK1 autophosphorylation and association with STAT2. Here, STAT2 antibody was used to detect endogenous protein. Arrowheads indicate JAK1-S (~130 kDa). (C) ISRE-luciferase reporter assay to assess vIRF-1 inhibition of TYK2-mediated signal transduction in 293T cells cotransfected with TYK2-expression and reporter plasmids and either vIRF-1 (virf1) or empty (-) expression vectors. Average values from duplicate samples for each condition are shown; error bars indicate standard deviations from the means. Statistical significance (P) was determined by student t-test (two-tailed, unpaired). (D) IFNAR1-S-based coprecipitation assay to test the influence of vIRF-1 (virf1), relative to empty-vector (-) transfection, on STAT2-receptor association, following IFNβ stimulation for 30 min. STAT2-CBD vector cotransfection provided expression of STAT2 above endogenous levels, to facilitate detection. (E) Effect of vIRF-1 on IFNβ receptor (IFNAR1) activation and association with TYK2. Transfectants expressing vIRF-1 or containing empty vector (-, negative control) and expressing, or lacking (-), introduced IFNAR1-CBD were left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h; TYK2-S was expressed in a subset of the transfected cultures. Cell lysates were analyzed for expression of the introduced proteins, and IFNAR1-CBD was affinity-precipitated from a subset of lysates to assess interaction of the receptor with TYK2 in response to vIRF-1. The numbers below the CBD blots show relative levels (-/+ vIRF-1) of IFNβ-induced lower IFNAR1 band (arrowheads) to total IFNAR1 (top plus bottom bands) from TYK2-overexpressing transfectants (+TYK2) and those devoid of TYK2 expression plasmid (-TYK2); values in the absence (-) of vIRF-1 are set at 1. For all precipitations (panels A, B, D and E), cultures were treated with DSP (2 mM, 30 min.) immediately prior to cell harvest, to stabilize targeted complexes.
    Figure Legend Snippet: Physical and functional interactions of vIRF-1 with JAKs. (A) TYK2-S and STAT2-CBD were expressed with (virf1) or without (-) vIRF-1 in transfected 293T cells. Cell lysates and S-protein affinity-precipitates were assessed for input protein expression and sedimentation by immunoblotting with CBD (STAT2), vIRF-1, and S-tag (TYK2) antibodies. Affinity-precipitated TYK-2-S was probed with phospho-tyrosine (PY)-specific antibody to identify the active, autophosphorylated form of the kinase. Dotted lines indicate lane deletions from single membranes; the arrowhead and asterisk indicate CBD-specific (STAT2) band and remnant S-tag signal (after blot stripping), respectively. (B) An equivalent experiment was performed to assess vIRF-1 effects on JAK1 autophosphorylation and association with STAT2. Here, STAT2 antibody was used to detect endogenous protein. Arrowheads indicate JAK1-S (~130 kDa). (C) ISRE-luciferase reporter assay to assess vIRF-1 inhibition of TYK2-mediated signal transduction in 293T cells cotransfected with TYK2-expression and reporter plasmids and either vIRF-1 (virf1) or empty (-) expression vectors. Average values from duplicate samples for each condition are shown; error bars indicate standard deviations from the means. Statistical significance (P) was determined by student t-test (two-tailed, unpaired). (D) IFNAR1-S-based coprecipitation assay to test the influence of vIRF-1 (virf1), relative to empty-vector (-) transfection, on STAT2-receptor association, following IFNβ stimulation for 30 min. STAT2-CBD vector cotransfection provided expression of STAT2 above endogenous levels, to facilitate detection. (E) Effect of vIRF-1 on IFNβ receptor (IFNAR1) activation and association with TYK2. Transfectants expressing vIRF-1 or containing empty vector (-, negative control) and expressing, or lacking (-), introduced IFNAR1-CBD were left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h; TYK2-S was expressed in a subset of the transfected cultures. Cell lysates were analyzed for expression of the introduced proteins, and IFNAR1-CBD was affinity-precipitated from a subset of lysates to assess interaction of the receptor with TYK2 in response to vIRF-1. The numbers below the CBD blots show relative levels (-/+ vIRF-1) of IFNβ-induced lower IFNAR1 band (arrowheads) to total IFNAR1 (top plus bottom bands) from TYK2-overexpressing transfectants (+TYK2) and those devoid of TYK2 expression plasmid (-TYK2); values in the absence (-) of vIRF-1 are set at 1. For all precipitations (panels A, B, D and E), cultures were treated with DSP (2 mM, 30 min.) immediately prior to cell harvest, to stabilize targeted complexes.

    Techniques Used: Functional Assay, Transfection, Expressing, Sedimentation, Stripping, Luciferase, Reporter Assay, Inhibition, Transduction, Two Tailed Test, Plasmid Preparation, Cotransfection, Activation Assay, Negative Control

    2) Product Images from "Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins"

    Article Title: Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins

    Journal: Analytical chemistry

    doi: 10.1021/ac400949t

    Antibody/fused-tag experiments. Measurements of AFM binding forces for: (A) CBD versus anti-CBD antibody and (B) MBP versus anti-MBP antibody. These experiments were performed using I C (with both fused domains CBD and MBP, according to ) with
    Figure Legend Snippet: Antibody/fused-tag experiments. Measurements of AFM binding forces for: (A) CBD versus anti-CBD antibody and (B) MBP versus anti-MBP antibody. These experiments were performed using I C (with both fused domains CBD and MBP, according to ) with

    Techniques Used: Binding Assay

    3) Product Images from "Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding"

    Article Title: Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding

    Journal: Journal of Virology

    doi: 10.1128/JVI.00965-17

    Profolding activity of vIL-6. (A) HEK293T cells were cotransfected with vectors expressing a GFP-fused version of the NHK folding variant of α1-antitrypsin (NHK-GFP) and vIL-6 or with NHK-GFP expression plasmid and empty vector (vec). At 48 h posttransfection, cell lysates were generated using NP-40-containing buffer, material microcentrifuged, and pellet (insoluble [insol.]) and supernatant (soluble [sol.]) fractions were denatured, SDS-PAGE fractionated, and immunoblotted for detection of GFP and also vIL-6 (to confirm expression) and ER luminal soluble protein BiP (to ensure appropriate separation of soluble and pellet fractions). The relative amounts of soluble to insoluble NHK-GFP are shown in the chart. (B) An independent assay was undertaken using secreted HHV-8 glycoprotein L (gL), fused to CBD, as the readout for folding. CBD-tagged HHV-8 gH was coexpressed with gL-CBD to enable secretory trafficking, and either vIL-6 expression plasmid or empty vector (vec) was cotransfected into HEK293T cells with the glycoprotein expression plasmids. After 48 h, cells and media were harvested for preparation of lysates and chitin bead precipitates (for concentration of secreted gL-CBD). Cell lysates (1% of total) and media precipitates (10%) were analyzed by immunoblotting for detection of gL-CBD; gH, vIL-6, and β-actin (loading control) were also analyzed in the lysate fractions. (C) An experiment equivalent to that of panel A was carried out in native HEK293T cells or VKORC1v2- or UGGT1-knockout (KO) derivatives. CBD- and KDEL motif-tagged hIL-6 (h6-C′-K) was included as a negative control along with empty vector (vec) for comparison with vIL-6 (CBD-fused, v6-CBD). Calculated ratios of NHK-GFP in the soluble and insoluble fractions of cell lysates are shown below each corresponding set of immunoblots. Digitally captured data from all immunoblots (panels A to C) were quantified using GeneTools (Syngene) analysis software.
    Figure Legend Snippet: Profolding activity of vIL-6. (A) HEK293T cells were cotransfected with vectors expressing a GFP-fused version of the NHK folding variant of α1-antitrypsin (NHK-GFP) and vIL-6 or with NHK-GFP expression plasmid and empty vector (vec). At 48 h posttransfection, cell lysates were generated using NP-40-containing buffer, material microcentrifuged, and pellet (insoluble [insol.]) and supernatant (soluble [sol.]) fractions were denatured, SDS-PAGE fractionated, and immunoblotted for detection of GFP and also vIL-6 (to confirm expression) and ER luminal soluble protein BiP (to ensure appropriate separation of soluble and pellet fractions). The relative amounts of soluble to insoluble NHK-GFP are shown in the chart. (B) An independent assay was undertaken using secreted HHV-8 glycoprotein L (gL), fused to CBD, as the readout for folding. CBD-tagged HHV-8 gH was coexpressed with gL-CBD to enable secretory trafficking, and either vIL-6 expression plasmid or empty vector (vec) was cotransfected into HEK293T cells with the glycoprotein expression plasmids. After 48 h, cells and media were harvested for preparation of lysates and chitin bead precipitates (for concentration of secreted gL-CBD). Cell lysates (1% of total) and media precipitates (10%) were analyzed by immunoblotting for detection of gL-CBD; gH, vIL-6, and β-actin (loading control) were also analyzed in the lysate fractions. (C) An experiment equivalent to that of panel A was carried out in native HEK293T cells or VKORC1v2- or UGGT1-knockout (KO) derivatives. CBD- and KDEL motif-tagged hIL-6 (h6-C′-K) was included as a negative control along with empty vector (vec) for comparison with vIL-6 (CBD-fused, v6-CBD). Calculated ratios of NHK-GFP in the soluble and insoluble fractions of cell lysates are shown below each corresponding set of immunoblots. Digitally captured data from all immunoblots (panels A to C) were quantified using GeneTools (Syngene) analysis software.

    Techniques Used: Activity Assay, Expressing, Variant Assay, Plasmid Preparation, Generated, SDS Page, Concentration Assay, Knock-Out, Negative Control, Western Blot, Software

    Interactions of vIL-6 and VKORC1v2 with GlucIIα and UGGT1. (A) Coprecipitation assays were carried out using lysates of cells cotransfected with expression plasmids for CBD-fused vIL-6 and S-tag-fused GlucIIα or CBD-fused VKORC1v2 (v2-CBD) and StrepII-tagged UGGT1 to test the interactions between these protein pairs. CBD- and KDEL ER retention motif-linked hIL-6 (hIL-6-CBD.K) and CBD-tagged VKORC1v1 (v1-CBD) were used as negative controls in the respective experiments. Protein complexes were precipitated with CBD-binding chitin beads, and SDS-PAGE-fractionated and membrane-transferred proteins in precipitates and cell lysates were identified by immunoblotting with CBD antibody to visualize “baits” vIL-6 and VKORC1v2 or with S-peptide or StrepII antibody for detection of their candidate binding partners. β-actin probing of cell lysates provided a loading control. (B) Further precipitations were undertaken to test whether vIL-6 and VKORC1v2 could interact with UGGT1 and GlucIIα, respectively. In this experiment, GlucIIα-S and StrepII-UGGT1 were coexpressed with CBD-tagged vIL-6 or VKORC1v2, or negative controls hIL-6-CBD.K (hIL-6-C′.K) or VKORC1v1-CBD (v1-CBD). Chitin bead precipitates and cell lysates were analyzed as described previously. (C) Immunoprecipitation (IP) of vIL-6 from BCBL-1 PEL cell lysates and subsequent immunoblotting for GlucIIα and UGGT1. A sample of the lysate (10% of amount used for IP) was analyzed alongside material precipitated with vIL-6 antiserum (vIL-6) or control rabbit serum.
    Figure Legend Snippet: Interactions of vIL-6 and VKORC1v2 with GlucIIα and UGGT1. (A) Coprecipitation assays were carried out using lysates of cells cotransfected with expression plasmids for CBD-fused vIL-6 and S-tag-fused GlucIIα or CBD-fused VKORC1v2 (v2-CBD) and StrepII-tagged UGGT1 to test the interactions between these protein pairs. CBD- and KDEL ER retention motif-linked hIL-6 (hIL-6-CBD.K) and CBD-tagged VKORC1v1 (v1-CBD) were used as negative controls in the respective experiments. Protein complexes were precipitated with CBD-binding chitin beads, and SDS-PAGE-fractionated and membrane-transferred proteins in precipitates and cell lysates were identified by immunoblotting with CBD antibody to visualize “baits” vIL-6 and VKORC1v2 or with S-peptide or StrepII antibody for detection of their candidate binding partners. β-actin probing of cell lysates provided a loading control. (B) Further precipitations were undertaken to test whether vIL-6 and VKORC1v2 could interact with UGGT1 and GlucIIα, respectively. In this experiment, GlucIIα-S and StrepII-UGGT1 were coexpressed with CBD-tagged vIL-6 or VKORC1v2, or negative controls hIL-6-CBD.K (hIL-6-C′.K) or VKORC1v1-CBD (v1-CBD). Chitin bead precipitates and cell lysates were analyzed as described previously. (C) Immunoprecipitation (IP) of vIL-6 from BCBL-1 PEL cell lysates and subsequent immunoblotting for GlucIIα and UGGT1. A sample of the lysate (10% of amount used for IP) was analyzed alongside material precipitated with vIL-6 antiserum (vIL-6) or control rabbit serum.

    Techniques Used: Expressing, Binding Assay, SDS Page, Immunoprecipitation

    4) Product Images from "Insulin-Like Growth Factor 2 Receptor Expression Is Promoted by Human Herpesvirus 8-Encoded Interleukin-6 and Contributes to Viral Latency and Productive Replication"

    Article Title: Insulin-Like Growth Factor 2 Receptor Expression Is Promoted by Human Herpesvirus 8-Encoded Interleukin-6 and Contributes to Viral Latency and Productive Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.02026-18

    Competitive vIL-6 and IGF2R interactions with VKORC1v2. (A) VKORC1v2-CBD (v2-CBD)-based coprecipitation assays were carried out in the absence or presence of vIL-6 coexpression to assess the influence of vIL-6 on VKORC1v2-IGF2R interaction. Chitin-bead
    Figure Legend Snippet: Competitive vIL-6 and IGF2R interactions with VKORC1v2. (A) VKORC1v2-CBD (v2-CBD)-based coprecipitation assays were carried out in the absence or presence of vIL-6 coexpression to assess the influence of vIL-6 on VKORC1v2-IGF2R interaction. Chitin-bead

    Techniques Used:

    Regulation of IGF2R expression by VKORC1v2. (A) Expression of IGF2R in HEK293T cell was monitored as a function of the dose of transfected VKORC1v2-CBD (v2-CBD) expression plasmid (0.4, 0.8, and 1.2 μg) and encoded protein. IGF2R and VKORC1v2-CBD
    Figure Legend Snippet: Regulation of IGF2R expression by VKORC1v2. (A) Expression of IGF2R in HEK293T cell was monitored as a function of the dose of transfected VKORC1v2-CBD (v2-CBD) expression plasmid (0.4, 0.8, and 1.2 μg) and encoded protein. IGF2R and VKORC1v2-CBD

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    Interaction between VKORC1v2 and IGF2R. (A) Coprecipitation assays were carried out using transfected HEK293T cell lysates as a source for affinity-tagged VKORC1v2 (CBD-fused, v2-CBD), or negative-control VKORC1v1-CBD (v1-CBD), and IGF2R (S-peptide-linked,
    Figure Legend Snippet: Interaction between VKORC1v2 and IGF2R. (A) Coprecipitation assays were carried out using transfected HEK293T cell lysates as a source for affinity-tagged VKORC1v2 (CBD-fused, v2-CBD), or negative-control VKORC1v1-CBD (v1-CBD), and IGF2R (S-peptide-linked,

    Techniques Used: Transfection, Negative Control

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    • anti cbd monoclonal antibody  (New England Biolabs)
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    New England Biolabs anti cbd monoclonal antibody
    Effects of vIRF-1 on complexing of ISGF3 components. (A-C) Flag-tagged STAT1 (A) or IRF9 (C) or <t>CBD-tagged</t> STAT2 (B) were expressed in 293T cells transfected with the respective expression plasmids and either vIRF-1 (virf1) or empty (-) expression vector; replicates were either left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h. Flag/CBD-tagged proteins were then immuno/affinity-precipitated from cell lysates, and coprecipitated ISGF3 proteins were detected by immunoblotting. Diagrams below the panels illustrate the main findings from each experiment. (D-E) Serial coprecipitations of STAT1 and STAT2 (D), STAT1 and IRF9 (E), and IRF9 and STAT2 (F), respectively Flag- and CBD-tagged, from lysates of transfected 293T cells expressing vIRF-1 (virf1) or cotransfected with empty vector (-). All transfectants were treated with IFNβ for 24 h prior to harvesting. First (Flag IP) and second (CBD AP) precipitates (Precip. 1, Precip. 2) were analyzed by immunoblotting for the tagged “bait” proteins, the third (endogenous) ISGF3 protein (including phosphorylated and total STAT1 and STAT2), and vIRF-1; lysates were immunoblotted for expression of input proteins. Illustrated below each panel of blots are the main findings. (G) Disruption by vIRF-1 of STAT1-STAT2 complexes isolated by Flag-IP (STAT1) and CBD-AP (STAT2) from IFNβ-treated transfected 293T cells. Immunoprecipitated material from vIRF-1-Flag (virf1) or empty control (cntl) vector-transfected cells was applied in two concentrations (1x, 2x) to dual-precipitation-derived STAT1/STAT2 complexes, and then mixtures were subjected to re-precipitation with chitin beads (binding STAT2-CBD). STAT1 and vIRF-1 associated with re-precipitated STAT2-CBD were identified by immunoblotting. Relative levels of co-precipitated STAT1, normalized to affinity-sedimented STAT2, are shown below the STAT1 blot (cntl/1x value set at 1). (H) An equivalent experiment was carried out using GST-fused recombinant vIRF-1 (virf1) or GST (negative control) to challenge STAT1 interaction with STAT2 in STAT1/STAT2 hetero-complexes isolated by IP/AP dual precipitations from IFNβ-treated 293T cells. Endogenous IRF9 interaction with STAT1/2 and competition by vIRF-1 were also monitored. Relative levels and integrities of the recombinant proteins are shown in the Coomassie-stained gel (right); arrowheads indicate the positions of the full-length proteins.
    Anti Cbd Monoclonal Antibody, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cbd monoclonal antibody/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cbd monoclonal antibody - by Bioz Stars, 2022-09
    93/100 stars
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    Effects of vIRF-1 on complexing of ISGF3 components. (A-C) Flag-tagged STAT1 (A) or IRF9 (C) or CBD-tagged STAT2 (B) were expressed in 293T cells transfected with the respective expression plasmids and either vIRF-1 (virf1) or empty (-) expression vector; replicates were either left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h. Flag/CBD-tagged proteins were then immuno/affinity-precipitated from cell lysates, and coprecipitated ISGF3 proteins were detected by immunoblotting. Diagrams below the panels illustrate the main findings from each experiment. (D-E) Serial coprecipitations of STAT1 and STAT2 (D), STAT1 and IRF9 (E), and IRF9 and STAT2 (F), respectively Flag- and CBD-tagged, from lysates of transfected 293T cells expressing vIRF-1 (virf1) or cotransfected with empty vector (-). All transfectants were treated with IFNβ for 24 h prior to harvesting. First (Flag IP) and second (CBD AP) precipitates (Precip. 1, Precip. 2) were analyzed by immunoblotting for the tagged “bait” proteins, the third (endogenous) ISGF3 protein (including phosphorylated and total STAT1 and STAT2), and vIRF-1; lysates were immunoblotted for expression of input proteins. Illustrated below each panel of blots are the main findings. (G) Disruption by vIRF-1 of STAT1-STAT2 complexes isolated by Flag-IP (STAT1) and CBD-AP (STAT2) from IFNβ-treated transfected 293T cells. Immunoprecipitated material from vIRF-1-Flag (virf1) or empty control (cntl) vector-transfected cells was applied in two concentrations (1x, 2x) to dual-precipitation-derived STAT1/STAT2 complexes, and then mixtures were subjected to re-precipitation with chitin beads (binding STAT2-CBD). STAT1 and vIRF-1 associated with re-precipitated STAT2-CBD were identified by immunoblotting. Relative levels of co-precipitated STAT1, normalized to affinity-sedimented STAT2, are shown below the STAT1 blot (cntl/1x value set at 1). (H) An equivalent experiment was carried out using GST-fused recombinant vIRF-1 (virf1) or GST (negative control) to challenge STAT1 interaction with STAT2 in STAT1/STAT2 hetero-complexes isolated by IP/AP dual precipitations from IFNβ-treated 293T cells. Endogenous IRF9 interaction with STAT1/2 and competition by vIRF-1 were also monitored. Relative levels and integrities of the recombinant proteins are shown in the Coomassie-stained gel (right); arrowheads indicate the positions of the full-length proteins.

    Journal: PLoS Pathogens

    Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

    doi: 10.1371/journal.ppat.1010676

    Figure Lengend Snippet: Effects of vIRF-1 on complexing of ISGF3 components. (A-C) Flag-tagged STAT1 (A) or IRF9 (C) or CBD-tagged STAT2 (B) were expressed in 293T cells transfected with the respective expression plasmids and either vIRF-1 (virf1) or empty (-) expression vector; replicates were either left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h. Flag/CBD-tagged proteins were then immuno/affinity-precipitated from cell lysates, and coprecipitated ISGF3 proteins were detected by immunoblotting. Diagrams below the panels illustrate the main findings from each experiment. (D-E) Serial coprecipitations of STAT1 and STAT2 (D), STAT1 and IRF9 (E), and IRF9 and STAT2 (F), respectively Flag- and CBD-tagged, from lysates of transfected 293T cells expressing vIRF-1 (virf1) or cotransfected with empty vector (-). All transfectants were treated with IFNβ for 24 h prior to harvesting. First (Flag IP) and second (CBD AP) precipitates (Precip. 1, Precip. 2) were analyzed by immunoblotting for the tagged “bait” proteins, the third (endogenous) ISGF3 protein (including phosphorylated and total STAT1 and STAT2), and vIRF-1; lysates were immunoblotted for expression of input proteins. Illustrated below each panel of blots are the main findings. (G) Disruption by vIRF-1 of STAT1-STAT2 complexes isolated by Flag-IP (STAT1) and CBD-AP (STAT2) from IFNβ-treated transfected 293T cells. Immunoprecipitated material from vIRF-1-Flag (virf1) or empty control (cntl) vector-transfected cells was applied in two concentrations (1x, 2x) to dual-precipitation-derived STAT1/STAT2 complexes, and then mixtures were subjected to re-precipitation with chitin beads (binding STAT2-CBD). STAT1 and vIRF-1 associated with re-precipitated STAT2-CBD were identified by immunoblotting. Relative levels of co-precipitated STAT1, normalized to affinity-sedimented STAT2, are shown below the STAT1 blot (cntl/1x value set at 1). (H) An equivalent experiment was carried out using GST-fused recombinant vIRF-1 (virf1) or GST (negative control) to challenge STAT1 interaction with STAT2 in STAT1/STAT2 hetero-complexes isolated by IP/AP dual precipitations from IFNβ-treated 293T cells. Endogenous IRF9 interaction with STAT1/2 and competition by vIRF-1 were also monitored. Relative levels and integrities of the recombinant proteins are shown in the Coomassie-stained gel (right); arrowheads indicate the positions of the full-length proteins.

    Article Snippet: Primary antibodies used for immunoblotting were: S (Abcam, catalog number ab184223); CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); STAT1, pSTAT1, IRF9, His6, GST and p53 from Santa Cruz Biotechnologies (catalog numbers sc464, sc-365893, sc-8394, sc-803, sc-138, and sc-126, respectively); STAT2 and pSTAT2 from Cell Signaling Technology (catalog numbers 72604 and 4441).

    Techniques: Transfection, Expressing, Plasmid Preparation, Isolation, Immunoprecipitation, Derivative Assay, Binding Assay, Recombinant, Negative Control, Staining

    Physical and functional interactions of vIRF-1 with JAKs. (A) TYK2-S and STAT2-CBD were expressed with (virf1) or without (-) vIRF-1 in transfected 293T cells. Cell lysates and S-protein affinity-precipitates were assessed for input protein expression and sedimentation by immunoblotting with CBD (STAT2), vIRF-1, and S-tag (TYK2) antibodies. Affinity-precipitated TYK-2-S was probed with phospho-tyrosine (PY)-specific antibody to identify the active, autophosphorylated form of the kinase. Dotted lines indicate lane deletions from single membranes; the arrowhead and asterisk indicate CBD-specific (STAT2) band and remnant S-tag signal (after blot stripping), respectively. (B) An equivalent experiment was performed to assess vIRF-1 effects on JAK1 autophosphorylation and association with STAT2. Here, STAT2 antibody was used to detect endogenous protein. Arrowheads indicate JAK1-S (~130 kDa). (C) ISRE-luciferase reporter assay to assess vIRF-1 inhibition of TYK2-mediated signal transduction in 293T cells cotransfected with TYK2-expression and reporter plasmids and either vIRF-1 (virf1) or empty (-) expression vectors. Average values from duplicate samples for each condition are shown; error bars indicate standard deviations from the means. Statistical significance (P) was determined by student t-test (two-tailed, unpaired). (D) IFNAR1-S-based coprecipitation assay to test the influence of vIRF-1 (virf1), relative to empty-vector (-) transfection, on STAT2-receptor association, following IFNβ stimulation for 30 min. STAT2-CBD vector cotransfection provided expression of STAT2 above endogenous levels, to facilitate detection. (E) Effect of vIRF-1 on IFNβ receptor (IFNAR1) activation and association with TYK2. Transfectants expressing vIRF-1 or containing empty vector (-, negative control) and expressing, or lacking (-), introduced IFNAR1-CBD were left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h; TYK2-S was expressed in a subset of the transfected cultures. Cell lysates were analyzed for expression of the introduced proteins, and IFNAR1-CBD was affinity-precipitated from a subset of lysates to assess interaction of the receptor with TYK2 in response to vIRF-1. The numbers below the CBD blots show relative levels (-/+ vIRF-1) of IFNβ-induced lower IFNAR1 band (arrowheads) to total IFNAR1 (top plus bottom bands) from TYK2-overexpressing transfectants (+TYK2) and those devoid of TYK2 expression plasmid (-TYK2); values in the absence (-) of vIRF-1 are set at 1. For all precipitations (panels A, B, D and E), cultures were treated with DSP (2 mM, 30 min.) immediately prior to cell harvest, to stabilize targeted complexes.

    Journal: PLoS Pathogens

    Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

    doi: 10.1371/journal.ppat.1010676

    Figure Lengend Snippet: Physical and functional interactions of vIRF-1 with JAKs. (A) TYK2-S and STAT2-CBD were expressed with (virf1) or without (-) vIRF-1 in transfected 293T cells. Cell lysates and S-protein affinity-precipitates were assessed for input protein expression and sedimentation by immunoblotting with CBD (STAT2), vIRF-1, and S-tag (TYK2) antibodies. Affinity-precipitated TYK-2-S was probed with phospho-tyrosine (PY)-specific antibody to identify the active, autophosphorylated form of the kinase. Dotted lines indicate lane deletions from single membranes; the arrowhead and asterisk indicate CBD-specific (STAT2) band and remnant S-tag signal (after blot stripping), respectively. (B) An equivalent experiment was performed to assess vIRF-1 effects on JAK1 autophosphorylation and association with STAT2. Here, STAT2 antibody was used to detect endogenous protein. Arrowheads indicate JAK1-S (~130 kDa). (C) ISRE-luciferase reporter assay to assess vIRF-1 inhibition of TYK2-mediated signal transduction in 293T cells cotransfected with TYK2-expression and reporter plasmids and either vIRF-1 (virf1) or empty (-) expression vectors. Average values from duplicate samples for each condition are shown; error bars indicate standard deviations from the means. Statistical significance (P) was determined by student t-test (two-tailed, unpaired). (D) IFNAR1-S-based coprecipitation assay to test the influence of vIRF-1 (virf1), relative to empty-vector (-) transfection, on STAT2-receptor association, following IFNβ stimulation for 30 min. STAT2-CBD vector cotransfection provided expression of STAT2 above endogenous levels, to facilitate detection. (E) Effect of vIRF-1 on IFNβ receptor (IFNAR1) activation and association with TYK2. Transfectants expressing vIRF-1 or containing empty vector (-, negative control) and expressing, or lacking (-), introduced IFNAR1-CBD were left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h; TYK2-S was expressed in a subset of the transfected cultures. Cell lysates were analyzed for expression of the introduced proteins, and IFNAR1-CBD was affinity-precipitated from a subset of lysates to assess interaction of the receptor with TYK2 in response to vIRF-1. The numbers below the CBD blots show relative levels (-/+ vIRF-1) of IFNβ-induced lower IFNAR1 band (arrowheads) to total IFNAR1 (top plus bottom bands) from TYK2-overexpressing transfectants (+TYK2) and those devoid of TYK2 expression plasmid (-TYK2); values in the absence (-) of vIRF-1 are set at 1. For all precipitations (panels A, B, D and E), cultures were treated with DSP (2 mM, 30 min.) immediately prior to cell harvest, to stabilize targeted complexes.

    Article Snippet: Primary antibodies used for immunoblotting were: S (Abcam, catalog number ab184223); CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); STAT1, pSTAT1, IRF9, His6, GST and p53 from Santa Cruz Biotechnologies (catalog numbers sc464, sc-365893, sc-8394, sc-803, sc-138, and sc-126, respectively); STAT2 and pSTAT2 from Cell Signaling Technology (catalog numbers 72604 and 4441).

    Techniques: Functional Assay, Transfection, Expressing, Sedimentation, Stripping, Luciferase, Reporter Assay, Inhibition, Transduction, Two Tailed Test, Plasmid Preparation, Cotransfection, Activation Assay, Negative Control

    Antibody/fused-tag experiments. Measurements of AFM binding forces for: (A) CBD versus anti-CBD antibody and (B) MBP versus anti-MBP antibody. These experiments were performed using I C (with both fused domains CBD and MBP, according to ) with

    Journal: Analytical chemistry

    Article Title: Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins

    doi: 10.1021/ac400949t

    Figure Lengend Snippet: Antibody/fused-tag experiments. Measurements of AFM binding forces for: (A) CBD versus anti-CBD antibody and (B) MBP versus anti-MBP antibody. These experiments were performed using I C (with both fused domains CBD and MBP, according to ) with

    Article Snippet: Anti-MBP and anti-CBD monoclonal antibodies were used for protein immobilization (New England Biolabs, Ipswich, MA).

    Techniques: Binding Assay

    Profolding activity of vIL-6. (A) HEK293T cells were cotransfected with vectors expressing a GFP-fused version of the NHK folding variant of α1-antitrypsin (NHK-GFP) and vIL-6 or with NHK-GFP expression plasmid and empty vector (vec). At 48 h posttransfection, cell lysates were generated using NP-40-containing buffer, material microcentrifuged, and pellet (insoluble [insol.]) and supernatant (soluble [sol.]) fractions were denatured, SDS-PAGE fractionated, and immunoblotted for detection of GFP and also vIL-6 (to confirm expression) and ER luminal soluble protein BiP (to ensure appropriate separation of soluble and pellet fractions). The relative amounts of soluble to insoluble NHK-GFP are shown in the chart. (B) An independent assay was undertaken using secreted HHV-8 glycoprotein L (gL), fused to CBD, as the readout for folding. CBD-tagged HHV-8 gH was coexpressed with gL-CBD to enable secretory trafficking, and either vIL-6 expression plasmid or empty vector (vec) was cotransfected into HEK293T cells with the glycoprotein expression plasmids. After 48 h, cells and media were harvested for preparation of lysates and chitin bead precipitates (for concentration of secreted gL-CBD). Cell lysates (1% of total) and media precipitates (10%) were analyzed by immunoblotting for detection of gL-CBD; gH, vIL-6, and β-actin (loading control) were also analyzed in the lysate fractions. (C) An experiment equivalent to that of panel A was carried out in native HEK293T cells or VKORC1v2- or UGGT1-knockout (KO) derivatives. CBD- and KDEL motif-tagged hIL-6 (h6-C′-K) was included as a negative control along with empty vector (vec) for comparison with vIL-6 (CBD-fused, v6-CBD). Calculated ratios of NHK-GFP in the soluble and insoluble fractions of cell lysates are shown below each corresponding set of immunoblots. Digitally captured data from all immunoblots (panels A to C) were quantified using GeneTools (Syngene) analysis software.

    Journal: Journal of Virology

    Article Title: Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding

    doi: 10.1128/JVI.00965-17

    Figure Lengend Snippet: Profolding activity of vIL-6. (A) HEK293T cells were cotransfected with vectors expressing a GFP-fused version of the NHK folding variant of α1-antitrypsin (NHK-GFP) and vIL-6 or with NHK-GFP expression plasmid and empty vector (vec). At 48 h posttransfection, cell lysates were generated using NP-40-containing buffer, material microcentrifuged, and pellet (insoluble [insol.]) and supernatant (soluble [sol.]) fractions were denatured, SDS-PAGE fractionated, and immunoblotted for detection of GFP and also vIL-6 (to confirm expression) and ER luminal soluble protein BiP (to ensure appropriate separation of soluble and pellet fractions). The relative amounts of soluble to insoluble NHK-GFP are shown in the chart. (B) An independent assay was undertaken using secreted HHV-8 glycoprotein L (gL), fused to CBD, as the readout for folding. CBD-tagged HHV-8 gH was coexpressed with gL-CBD to enable secretory trafficking, and either vIL-6 expression plasmid or empty vector (vec) was cotransfected into HEK293T cells with the glycoprotein expression plasmids. After 48 h, cells and media were harvested for preparation of lysates and chitin bead precipitates (for concentration of secreted gL-CBD). Cell lysates (1% of total) and media precipitates (10%) were analyzed by immunoblotting for detection of gL-CBD; gH, vIL-6, and β-actin (loading control) were also analyzed in the lysate fractions. (C) An experiment equivalent to that of panel A was carried out in native HEK293T cells or VKORC1v2- or UGGT1-knockout (KO) derivatives. CBD- and KDEL motif-tagged hIL-6 (h6-C′-K) was included as a negative control along with empty vector (vec) for comparison with vIL-6 (CBD-fused, v6-CBD). Calculated ratios of NHK-GFP in the soluble and insoluble fractions of cell lysates are shown below each corresponding set of immunoblots. Digitally captured data from all immunoblots (panels A to C) were quantified using GeneTools (Syngene) analysis software.

    Article Snippet: Commercially acquired antibodies used for immunoblotting and immunoprecipitation were as follows: CBD (New England BioLabs, catalog no. E8034S), S-tag (Cell Signaling Technology, catalog no. 8476S), GFP (Santa Cruz Biotechnology, catalog no. sc-8334), Strep II (Qiagen, catalog no. 34850), Flag (Sigma, catalog no. F7425), hemagglutinin (HA; Sigma, catalog no. H6908), β-actin (Sigma, catalog no. A5316), BiP (BD Biosciences, catalog number 610978), STAT3 (Santa Cruz Biotechnology, catalog number sc-482), pSTAT3 (Cell Signaling Technologies, catalog number 9131S), and gp130 (Santa Cruz Biotechnology, catalog number sc-655).

    Techniques: Activity Assay, Expressing, Variant Assay, Plasmid Preparation, Generated, SDS Page, Concentration Assay, Knock-Out, Negative Control, Western Blot, Software

    Interactions of vIL-6 and VKORC1v2 with GlucIIα and UGGT1. (A) Coprecipitation assays were carried out using lysates of cells cotransfected with expression plasmids for CBD-fused vIL-6 and S-tag-fused GlucIIα or CBD-fused VKORC1v2 (v2-CBD) and StrepII-tagged UGGT1 to test the interactions between these protein pairs. CBD- and KDEL ER retention motif-linked hIL-6 (hIL-6-CBD.K) and CBD-tagged VKORC1v1 (v1-CBD) were used as negative controls in the respective experiments. Protein complexes were precipitated with CBD-binding chitin beads, and SDS-PAGE-fractionated and membrane-transferred proteins in precipitates and cell lysates were identified by immunoblotting with CBD antibody to visualize “baits” vIL-6 and VKORC1v2 or with S-peptide or StrepII antibody for detection of their candidate binding partners. β-actin probing of cell lysates provided a loading control. (B) Further precipitations were undertaken to test whether vIL-6 and VKORC1v2 could interact with UGGT1 and GlucIIα, respectively. In this experiment, GlucIIα-S and StrepII-UGGT1 were coexpressed with CBD-tagged vIL-6 or VKORC1v2, or negative controls hIL-6-CBD.K (hIL-6-C′.K) or VKORC1v1-CBD (v1-CBD). Chitin bead precipitates and cell lysates were analyzed as described previously. (C) Immunoprecipitation (IP) of vIL-6 from BCBL-1 PEL cell lysates and subsequent immunoblotting for GlucIIα and UGGT1. A sample of the lysate (10% of amount used for IP) was analyzed alongside material precipitated with vIL-6 antiserum (vIL-6) or control rabbit serum.

    Journal: Journal of Virology

    Article Title: Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding

    doi: 10.1128/JVI.00965-17

    Figure Lengend Snippet: Interactions of vIL-6 and VKORC1v2 with GlucIIα and UGGT1. (A) Coprecipitation assays were carried out using lysates of cells cotransfected with expression plasmids for CBD-fused vIL-6 and S-tag-fused GlucIIα or CBD-fused VKORC1v2 (v2-CBD) and StrepII-tagged UGGT1 to test the interactions between these protein pairs. CBD- and KDEL ER retention motif-linked hIL-6 (hIL-6-CBD.K) and CBD-tagged VKORC1v1 (v1-CBD) were used as negative controls in the respective experiments. Protein complexes were precipitated with CBD-binding chitin beads, and SDS-PAGE-fractionated and membrane-transferred proteins in precipitates and cell lysates were identified by immunoblotting with CBD antibody to visualize “baits” vIL-6 and VKORC1v2 or with S-peptide or StrepII antibody for detection of their candidate binding partners. β-actin probing of cell lysates provided a loading control. (B) Further precipitations were undertaken to test whether vIL-6 and VKORC1v2 could interact with UGGT1 and GlucIIα, respectively. In this experiment, GlucIIα-S and StrepII-UGGT1 were coexpressed with CBD-tagged vIL-6 or VKORC1v2, or negative controls hIL-6-CBD.K (hIL-6-C′.K) or VKORC1v1-CBD (v1-CBD). Chitin bead precipitates and cell lysates were analyzed as described previously. (C) Immunoprecipitation (IP) of vIL-6 from BCBL-1 PEL cell lysates and subsequent immunoblotting for GlucIIα and UGGT1. A sample of the lysate (10% of amount used for IP) was analyzed alongside material precipitated with vIL-6 antiserum (vIL-6) or control rabbit serum.

    Article Snippet: Commercially acquired antibodies used for immunoblotting and immunoprecipitation were as follows: CBD (New England BioLabs, catalog no. E8034S), S-tag (Cell Signaling Technology, catalog no. 8476S), GFP (Santa Cruz Biotechnology, catalog no. sc-8334), Strep II (Qiagen, catalog no. 34850), Flag (Sigma, catalog no. F7425), hemagglutinin (HA; Sigma, catalog no. H6908), β-actin (Sigma, catalog no. A5316), BiP (BD Biosciences, catalog number 610978), STAT3 (Santa Cruz Biotechnology, catalog number sc-482), pSTAT3 (Cell Signaling Technologies, catalog number 9131S), and gp130 (Santa Cruz Biotechnology, catalog number sc-655).

    Techniques: Expressing, Binding Assay, SDS Page, Immunoprecipitation

    Competitive vIL-6 and IGF2R interactions with VKORC1v2. (A) VKORC1v2-CBD (v2-CBD)-based coprecipitation assays were carried out in the absence or presence of vIL-6 coexpression to assess the influence of vIL-6 on VKORC1v2-IGF2R interaction. Chitin-bead

    Journal: Journal of Virology

    Article Title: Insulin-Like Growth Factor 2 Receptor Expression Is Promoted by Human Herpesvirus 8-Encoded Interleukin-6 and Contributes to Viral Latency and Productive Replication

    doi: 10.1128/JVI.02026-18

    Figure Lengend Snippet: Competitive vIL-6 and IGF2R interactions with VKORC1v2. (A) VKORC1v2-CBD (v2-CBD)-based coprecipitation assays were carried out in the absence or presence of vIL-6 coexpression to assess the influence of vIL-6 on VKORC1v2-IGF2R interaction. Chitin-bead

    Article Snippet: Primary antibodies used in this study were directed to S-peptide (Abcam, catalog no. ab184223), CBD (New England BioLabs, catalog. no. E8034S), IGF2R (Abcam, catalog no. ab124767), β-actin (Sigma, catalog no. A5316), Flag (Sigma, catalog no. F1804), LANA (Advanced Biotechnologies, catalog no. 13-210-100), pSTAT3 (Cell Signaling Technology, catalog no. 9131S), and STAT3 (Santa Cruz, catalog no. sc-482).

    Techniques:

    Regulation of IGF2R expression by VKORC1v2. (A) Expression of IGF2R in HEK293T cell was monitored as a function of the dose of transfected VKORC1v2-CBD (v2-CBD) expression plasmid (0.4, 0.8, and 1.2 μg) and encoded protein. IGF2R and VKORC1v2-CBD

    Journal: Journal of Virology

    Article Title: Insulin-Like Growth Factor 2 Receptor Expression Is Promoted by Human Herpesvirus 8-Encoded Interleukin-6 and Contributes to Viral Latency and Productive Replication

    doi: 10.1128/JVI.02026-18

    Figure Lengend Snippet: Regulation of IGF2R expression by VKORC1v2. (A) Expression of IGF2R in HEK293T cell was monitored as a function of the dose of transfected VKORC1v2-CBD (v2-CBD) expression plasmid (0.4, 0.8, and 1.2 μg) and encoded protein. IGF2R and VKORC1v2-CBD

    Article Snippet: Primary antibodies used in this study were directed to S-peptide (Abcam, catalog no. ab184223), CBD (New England BioLabs, catalog. no. E8034S), IGF2R (Abcam, catalog no. ab124767), β-actin (Sigma, catalog no. A5316), Flag (Sigma, catalog no. F1804), LANA (Advanced Biotechnologies, catalog no. 13-210-100), pSTAT3 (Cell Signaling Technology, catalog no. 9131S), and STAT3 (Santa Cruz, catalog no. sc-482).

    Techniques: Expressing, Transfection, Plasmid Preparation

    Interaction between VKORC1v2 and IGF2R. (A) Coprecipitation assays were carried out using transfected HEK293T cell lysates as a source for affinity-tagged VKORC1v2 (CBD-fused, v2-CBD), or negative-control VKORC1v1-CBD (v1-CBD), and IGF2R (S-peptide-linked,

    Journal: Journal of Virology

    Article Title: Insulin-Like Growth Factor 2 Receptor Expression Is Promoted by Human Herpesvirus 8-Encoded Interleukin-6 and Contributes to Viral Latency and Productive Replication

    doi: 10.1128/JVI.02026-18

    Figure Lengend Snippet: Interaction between VKORC1v2 and IGF2R. (A) Coprecipitation assays were carried out using transfected HEK293T cell lysates as a source for affinity-tagged VKORC1v2 (CBD-fused, v2-CBD), or negative-control VKORC1v1-CBD (v1-CBD), and IGF2R (S-peptide-linked,

    Article Snippet: Primary antibodies used in this study were directed to S-peptide (Abcam, catalog no. ab184223), CBD (New England BioLabs, catalog. no. E8034S), IGF2R (Abcam, catalog no. ab124767), β-actin (Sigma, catalog no. A5316), Flag (Sigma, catalog no. F1804), LANA (Advanced Biotechnologies, catalog no. 13-210-100), pSTAT3 (Cell Signaling Technology, catalog no. 9131S), and STAT3 (Santa Cruz, catalog no. sc-482).

    Techniques: Transfection, Negative Control