goat anti mouse igg  (Jackson Immuno)

 
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    Name:
    Goat Anti Mouse IgG F ab 2 fragment specific
    Description:
    Polyclonal antisera from immunized hosts are lipid extracted to improve clarity salt fractionated dialyzed against phosphate buffered saline containing sodium azide and freeze dried Antisera against whole serums are obtained by immunizing host animals with whole serum Antisera against whole IgG molecules i e Anti IgG H L are recommended for bridging PAP to primary antibodies Based on immunoelectrophoresis the antibody reacts with the F ab 2 Fab portion of mouse IgG It also reacts with the light chains of other mouse immunoglobulins No antibody was detected against the Fc portion of mouse IgG or against non immunoglobulin serum proteins The antibody may cross react with immunogloublins from other species
    Catalog Number:
    115-001-006
    Price:
    66.0
    Category:
    Antisera With Limited Stock
    Conjugate:
    Unconjugated
    Size:
    2 0 ml
    Host:
    Goat
    Buy from Supplier


    Structured Review

    Jackson Immuno goat anti mouse igg
    Development of <t>IgG</t> and <t>IgM</t> autoantibodies against double-stranded DNA (anti-dsDNA) antibodies in double-deficient mice. In (A) , the results from 3- to 4-month-old toll-like receptor 9 (TLR9) × Dnase1l3 mice are displayed. The genotypes of the mice are denoted at the axis. In (B) , the results from 3-month-old FcgR2b × Dnase1l3 mice are displayed. The genotypes of the mice are denoted at the axis. The dotted lines represent anti-dsDNA antibody levels of a serum pool of 9-month-old female NZB/W mice. Data are presented as box plot with individual data represented by filled circles. (C) Follow-up of IgG anti-dsDNA autoantibody levels in individual female FcgR2b × Dnase1l3 double-deficient mice (red) and male FcgR2b yaa × Dnase1l3 double-deficient mice (blue). In (D) , the IgM anti-dsDNA levels from a cohort of 2- to 3-month-old FcgR2b × Dnase1l3 mice are displayed. The genotypes of the mice are denoted at the axis. Data are presented as box plot with individual data represented by filled circles (* p
    Polyclonal antisera from immunized hosts are lipid extracted to improve clarity salt fractionated dialyzed against phosphate buffered saline containing sodium azide and freeze dried Antisera against whole serums are obtained by immunizing host animals with whole serum Antisera against whole IgG molecules i e Anti IgG H L are recommended for bridging PAP to primary antibodies Based on immunoelectrophoresis the antibody reacts with the F ab 2 Fab portion of mouse IgG It also reacts with the light chains of other mouse immunoglobulins No antibody was detected against the Fc portion of mouse IgG or against non immunoglobulin serum proteins The antibody may cross react with immunogloublins from other species
    https://www.bioz.com/result/goat anti mouse igg/product/Jackson Immuno
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti mouse igg - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "Epistatic Interactions Between Mutations of Deoxyribonuclease 1-Like 3 and the Inhibitory Fc Gamma Receptor IIB Result in Very Early and Massive Autoantibodies Against Double-Stranded DNA"

    Article Title: Epistatic Interactions Between Mutations of Deoxyribonuclease 1-Like 3 and the Inhibitory Fc Gamma Receptor IIB Result in Very Early and Massive Autoantibodies Against Double-Stranded DNA

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01551

    Development of IgG and IgM autoantibodies against double-stranded DNA (anti-dsDNA) antibodies in double-deficient mice. In (A) , the results from 3- to 4-month-old toll-like receptor 9 (TLR9) × Dnase1l3 mice are displayed. The genotypes of the mice are denoted at the axis. In (B) , the results from 3-month-old FcgR2b × Dnase1l3 mice are displayed. The genotypes of the mice are denoted at the axis. The dotted lines represent anti-dsDNA antibody levels of a serum pool of 9-month-old female NZB/W mice. Data are presented as box plot with individual data represented by filled circles. (C) Follow-up of IgG anti-dsDNA autoantibody levels in individual female FcgR2b × Dnase1l3 double-deficient mice (red) and male FcgR2b yaa × Dnase1l3 double-deficient mice (blue). In (D) , the IgM anti-dsDNA levels from a cohort of 2- to 3-month-old FcgR2b × Dnase1l3 mice are displayed. The genotypes of the mice are denoted at the axis. Data are presented as box plot with individual data represented by filled circles (* p
    Figure Legend Snippet: Development of IgG and IgM autoantibodies against double-stranded DNA (anti-dsDNA) antibodies in double-deficient mice. In (A) , the results from 3- to 4-month-old toll-like receptor 9 (TLR9) × Dnase1l3 mice are displayed. The genotypes of the mice are denoted at the axis. In (B) , the results from 3-month-old FcgR2b × Dnase1l3 mice are displayed. The genotypes of the mice are denoted at the axis. The dotted lines represent anti-dsDNA antibody levels of a serum pool of 9-month-old female NZB/W mice. Data are presented as box plot with individual data represented by filled circles. (C) Follow-up of IgG anti-dsDNA autoantibody levels in individual female FcgR2b × Dnase1l3 double-deficient mice (red) and male FcgR2b yaa × Dnase1l3 double-deficient mice (blue). In (D) , the IgM anti-dsDNA levels from a cohort of 2- to 3-month-old FcgR2b × Dnase1l3 mice are displayed. The genotypes of the mice are denoted at the axis. Data are presented as box plot with individual data represented by filled circles (* p

    Techniques Used: Mouse Assay

    2) Product Images from "CD59 signaling and membrane pores drive Syk-dependent erythrocyte necroptosis"

    Article Title: CD59 signaling and membrane pores drive Syk-dependent erythrocyte necroptosis

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.135

    The combination of hCD59 crosslinking and PLY induces RBC necroptosis. ( a ) Hemolysis assay showing that mAb binding or crosslinking of hCD59 (CD59) or hCD59 binding or crosslinking with the binding domain of VLY fused to GFP (VLYD4-GFP) does not induce death of RBCs. When hCD59 is bound or crosslinked with ( b ) mAb against hCD59 or ( c ) VLYD4-GFP it results in an enhancement of RBC death by the hCD59-independent PLY (0.2 HU). Enhanced RBC death by PLY in the presence of crosslinked hCD59 is prevented by ( d ) the RIP1 inhibitor, nec-1 (50 μ M) or ( e ) the FasL neutralizing mAb, NOK-1 (1 μ g/ml). Enhanced RBC death by PLY in the presence of crosslinked hCD59 is reduced by inhibitors of downstream effector pathways in RBC necroptosis including ( f ) inhibition of ceramide formation with acid sphingomyelinase (aSMase) inhibitor desipramine (DPA, 20 μ M), ( g ) inhibition of reactive oxygen species (ROS) with the iron chelator 2,2 bipyridyl (bipyridyl, 100 μ M) or ( h ) inhibition of AGEs with pyridoxamine (1 mM). Time points for hemolysis assays were 30 min. When pharmacological or mAb inhibitors were used RBCs were treated for 1 h before the addition of stimuli. IgG=irrelevant IgG control, CD55=mAb against hCD55, GFP=addition of rGFP alone. Error bars in hemolysis assays represent standard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P
    Figure Legend Snippet: The combination of hCD59 crosslinking and PLY induces RBC necroptosis. ( a ) Hemolysis assay showing that mAb binding or crosslinking of hCD59 (CD59) or hCD59 binding or crosslinking with the binding domain of VLY fused to GFP (VLYD4-GFP) does not induce death of RBCs. When hCD59 is bound or crosslinked with ( b ) mAb against hCD59 or ( c ) VLYD4-GFP it results in an enhancement of RBC death by the hCD59-independent PLY (0.2 HU). Enhanced RBC death by PLY in the presence of crosslinked hCD59 is prevented by ( d ) the RIP1 inhibitor, nec-1 (50 μ M) or ( e ) the FasL neutralizing mAb, NOK-1 (1 μ g/ml). Enhanced RBC death by PLY in the presence of crosslinked hCD59 is reduced by inhibitors of downstream effector pathways in RBC necroptosis including ( f ) inhibition of ceramide formation with acid sphingomyelinase (aSMase) inhibitor desipramine (DPA, 20 μ M), ( g ) inhibition of reactive oxygen species (ROS) with the iron chelator 2,2 bipyridyl (bipyridyl, 100 μ M) or ( h ) inhibition of AGEs with pyridoxamine (1 mM). Time points for hemolysis assays were 30 min. When pharmacological or mAb inhibitors were used RBCs were treated for 1 h before the addition of stimuli. IgG=irrelevant IgG control, CD55=mAb against hCD55, GFP=addition of rGFP alone. Error bars in hemolysis assays represent standard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P

    Techniques Used: Hemolysis Assay, Binding Assay, Inhibition, Standard Deviation

    RBC necroptosis induced by hCD59 depends on membrane pore size and nature. When combined with the hCD59-independent cholesterol-dependent cytolysins (CDCs) ( a ) arcanolysin (ALN) or ( b ) listeriolysin O (LLO) or ( c ) the MAC of complement, hCD59 crosslinking (CL-CD59) induces robust RBC necroptosis as indicated by inhibition of RBC death with nec-1 (50 μ M). ALN, LLO, and the MAC all form protein-based pores ≥10 nm in diameter. ( d ) When combined with the hCD59-independent A-tox, which forms protein-based pores of 1–2 nm in diameter, induction of RBC necroptosis by hCD59 is weak. ( e ) When combined with β -hemolysin, which forms polyene-based pores, hCD59 crosslinking fails to induce RBC necroptosis. All PFTs were used at 0.2 HU. ( f ) hCD59 crosslinking does not induce RBC necroptosis when combined with two forms of eryptosis damage, hyperosmotic stress (osm) and excess calcium (cal). All hemolysis assays except for eryptosis were completed at a time point of 30 min. Eryptosis stimuli were added to RBCs over a period of 24 h. When Nec-1 was used, RBCs were treated for 1 h before the addition of stimuli. CL-IgG=crosslinked irrelevant IgG control. Error bars in hemolysis assays represent stand ard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P
    Figure Legend Snippet: RBC necroptosis induced by hCD59 depends on membrane pore size and nature. When combined with the hCD59-independent cholesterol-dependent cytolysins (CDCs) ( a ) arcanolysin (ALN) or ( b ) listeriolysin O (LLO) or ( c ) the MAC of complement, hCD59 crosslinking (CL-CD59) induces robust RBC necroptosis as indicated by inhibition of RBC death with nec-1 (50 μ M). ALN, LLO, and the MAC all form protein-based pores ≥10 nm in diameter. ( d ) When combined with the hCD59-independent A-tox, which forms protein-based pores of 1–2 nm in diameter, induction of RBC necroptosis by hCD59 is weak. ( e ) When combined with β -hemolysin, which forms polyene-based pores, hCD59 crosslinking fails to induce RBC necroptosis. All PFTs were used at 0.2 HU. ( f ) hCD59 crosslinking does not induce RBC necroptosis when combined with two forms of eryptosis damage, hyperosmotic stress (osm) and excess calcium (cal). All hemolysis assays except for eryptosis were completed at a time point of 30 min. Eryptosis stimuli were added to RBCs over a period of 24 h. When Nec-1 was used, RBCs were treated for 1 h before the addition of stimuli. CL-IgG=crosslinked irrelevant IgG control. Error bars in hemolysis assays represent stand ard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P

    Techniques Used: Inhibition

    Binding or crosslinking of the hCD59 receptor leads to FasL-dependent phosphorylation of RIP1 in RBCs. ( a ) RIP1 IPs showing p-RIP1 in response to binding of hCD59 by the specific mAb MEM-43 (CD59, 1 μ g/mL) or a His-tagged version of the binding domain of VLY (VLYD4, 100 ng/ml). Further crosslinking of hCD59 with an anti-mouse IgG pAb (CL-CD59) or an anti-His mAb (CL-VLYD4) leads to more robust levels of p-RIP1. ( b ) IPs similar to those in ( a ) showing p-RIP1 specifically in response to hCD59 binding. A version of PLY modified with the binding domain of VLY (PLY-VLYD4) induces p-RIP1 while VLY modified with the binding domain of PLY (VLY-PLYD4) does not. RIP1 is phosphorylated specifically in response to CL-CD59 or CL-VLYD4. Crosslinking of hCD55 (CL-CD55) or PLYD4 (CL-PLYD4) does not result in p-RIP1. The p-RIP1 induced by CL-CD59 is retained in combination with ( c ) the hCD59-independent PFTs, PLY or A-tox, or ( d ) the eryptosis stimuli, hyperosmotic stress (osm) or excess calcium (cal). ( e ) The p-RIP1 induced by CL-CD59 or CL-VLYD4 is prevented by the RIP1 inhibitor nec-1 (50 μ M) or neutralization of FasL (NOK-1, 1 μ g/ml). Treatments for all experiments except for eryptosis were 1 h. Eryptosis stimuli were added to RBCs for a period of 24 h. When nec-1 or mAb NOK-1 was used, RBCs were treated with these inhibitors for 1 h before the addition of stimuli
    Figure Legend Snippet: Binding or crosslinking of the hCD59 receptor leads to FasL-dependent phosphorylation of RIP1 in RBCs. ( a ) RIP1 IPs showing p-RIP1 in response to binding of hCD59 by the specific mAb MEM-43 (CD59, 1 μ g/mL) or a His-tagged version of the binding domain of VLY (VLYD4, 100 ng/ml). Further crosslinking of hCD59 with an anti-mouse IgG pAb (CL-CD59) or an anti-His mAb (CL-VLYD4) leads to more robust levels of p-RIP1. ( b ) IPs similar to those in ( a ) showing p-RIP1 specifically in response to hCD59 binding. A version of PLY modified with the binding domain of VLY (PLY-VLYD4) induces p-RIP1 while VLY modified with the binding domain of PLY (VLY-PLYD4) does not. RIP1 is phosphorylated specifically in response to CL-CD59 or CL-VLYD4. Crosslinking of hCD55 (CL-CD55) or PLYD4 (CL-PLYD4) does not result in p-RIP1. The p-RIP1 induced by CL-CD59 is retained in combination with ( c ) the hCD59-independent PFTs, PLY or A-tox, or ( d ) the eryptosis stimuli, hyperosmotic stress (osm) or excess calcium (cal). ( e ) The p-RIP1 induced by CL-CD59 or CL-VLYD4 is prevented by the RIP1 inhibitor nec-1 (50 μ M) or neutralization of FasL (NOK-1, 1 μ g/ml). Treatments for all experiments except for eryptosis were 1 h. Eryptosis stimuli were added to RBCs for a period of 24 h. When nec-1 or mAb NOK-1 was used, RBCs were treated with these inhibitors for 1 h before the addition of stimuli

    Techniques Used: Binding Assay, Modification, Neutralization

    Functional pore formation is necessary for hCD59-induced RBC necroptosis. ( a and b ) Hemolysis assays showing that osmotic protection with dextran (500 000 MW) prevents all RBC death by the RBC necroptosis stimuli VLY and ILY. ( c ) Toxoids of PLY (PdB) and A-tox (A-tox toxoid), which are defective for pore formation, do not induce RBC necroptosis when combined with hCD59 crosslinking (CL-CD59). RBC necroptosis induced by hCD59 crosslinking combined with ( d ) PLY, ( e ) ALN, ( f ) LLO, or ( g ) A-tox is completely prevented by osmoprotection with dextran. Time points for all hemolysis assays were 30 min. PLY, ALN, LLO, and A-tox were used at 0.2 HU. CL-IgG=crosslinked irrelevant IgG control. Error bars in hemolysis assays represent standard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P
    Figure Legend Snippet: Functional pore formation is necessary for hCD59-induced RBC necroptosis. ( a and b ) Hemolysis assays showing that osmotic protection with dextran (500 000 MW) prevents all RBC death by the RBC necroptosis stimuli VLY and ILY. ( c ) Toxoids of PLY (PdB) and A-tox (A-tox toxoid), which are defective for pore formation, do not induce RBC necroptosis when combined with hCD59 crosslinking (CL-CD59). RBC necroptosis induced by hCD59 crosslinking combined with ( d ) PLY, ( e ) ALN, ( f ) LLO, or ( g ) A-tox is completely prevented by osmoprotection with dextran. Time points for all hemolysis assays were 30 min. PLY, ALN, LLO, and A-tox were used at 0.2 HU. CL-IgG=crosslinked irrelevant IgG control. Error bars in hemolysis assays represent standard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P

    Techniques Used: Functional Assay, Standard Deviation

    The combination of hCD59 crosslinking and PLY induces RBC necroptosis. ( a ) Hemolysis assay showing that mAb binding or crosslinking of hCD59 (CD59) or hCD59 binding or crosslinking with the binding domain of VLY fused to GFP (VLYD4-GFP) does not induce death of RBCs. When hCD59 is bound or crosslinked with ( b ) mAb against hCD59 or ( c ) VLYD4-GFP it results in an enhancement of RBC death by the hCD59-independent PLY (0.2 HU). Enhanced RBC death by PLY in the presence of crosslinked hCD59 is prevented by ( d ) the RIP1 inhibitor, nec-1 (50 μ M) or ( e ) the FasL neutralizing mAb, NOK-1 (1 μ g/ml). Enhanced RBC death by PLY in the presence of crosslinked hCD59 is reduced by inhibitors of downstream effector pathways in RBC necroptosis including ( f ) inhibition of ceramide formation with acid sphingomyelinase (aSMase) inhibitor desipramine (DPA, 20 μ M), ( g ) inhibition of reactive oxygen species (ROS) with the iron chelator 2,2 bipyridyl (bipyridyl, 100 μ M) or ( h ) inhibition of AGEs with pyridoxamine (1 mM). Time points for hemolysis assays were 30 min. When pharmacological or mAb inhibitors were used RBCs were treated for 1 h before the addition of stimuli. IgG=irrelevant IgG control, CD55=mAb against hCD55, GFP=addition of rGFP alone. Error bars in hemolysis assays represent standard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P
    Figure Legend Snippet: The combination of hCD59 crosslinking and PLY induces RBC necroptosis. ( a ) Hemolysis assay showing that mAb binding or crosslinking of hCD59 (CD59) or hCD59 binding or crosslinking with the binding domain of VLY fused to GFP (VLYD4-GFP) does not induce death of RBCs. When hCD59 is bound or crosslinked with ( b ) mAb against hCD59 or ( c ) VLYD4-GFP it results in an enhancement of RBC death by the hCD59-independent PLY (0.2 HU). Enhanced RBC death by PLY in the presence of crosslinked hCD59 is prevented by ( d ) the RIP1 inhibitor, nec-1 (50 μ M) or ( e ) the FasL neutralizing mAb, NOK-1 (1 μ g/ml). Enhanced RBC death by PLY in the presence of crosslinked hCD59 is reduced by inhibitors of downstream effector pathways in RBC necroptosis including ( f ) inhibition of ceramide formation with acid sphingomyelinase (aSMase) inhibitor desipramine (DPA, 20 μ M), ( g ) inhibition of reactive oxygen species (ROS) with the iron chelator 2,2 bipyridyl (bipyridyl, 100 μ M) or ( h ) inhibition of AGEs with pyridoxamine (1 mM). Time points for hemolysis assays were 30 min. When pharmacological or mAb inhibitors were used RBCs were treated for 1 h before the addition of stimuli. IgG=irrelevant IgG control, CD55=mAb against hCD55, GFP=addition of rGFP alone. Error bars in hemolysis assays represent standard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P

    Techniques Used: Hemolysis Assay, Binding Assay, Inhibition, Standard Deviation

    RBC necroptosis induced by hCD59 depends on membrane pore size and nature. When combined with the hCD59-independent cholesterol-dependent cytolysins (CDCs) ( a ) arcanolysin (ALN) or ( b ) listeriolysin O (LLO) or ( c ) the MAC of complement, hCD59 crosslinking (CL-CD59) induces robust RBC necroptosis as indicated by inhibition of RBC death with nec-1 (50 μ M). ALN, LLO, and the MAC all form protein-based pores ≥10 nm in diameter. ( d ) When combined with the hCD59-independent A-tox, which forms protein-based pores of 1–2 nm in diameter, induction of RBC necroptosis by hCD59 is weak. ( e ) When combined with β -hemolysin, which forms polyene-based pores, hCD59 crosslinking fails to induce RBC necroptosis. All PFTs were used at 0.2 HU. ( f ) hCD59 crosslinking does not induce RBC necroptosis when combined with two forms of eryptosis damage, hyperosmotic stress (osm) and excess calcium (cal). All hemolysis assays except for eryptosis were completed at a time point of 30 min. Eryptosis stimuli were added to RBCs over a period of 24 h. When Nec-1 was used, RBCs were treated for 1 h before the addition of stimuli. CL-IgG=crosslinked irrelevant IgG control. Error bars in hemolysis assays represent stand ard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P
    Figure Legend Snippet: RBC necroptosis induced by hCD59 depends on membrane pore size and nature. When combined with the hCD59-independent cholesterol-dependent cytolysins (CDCs) ( a ) arcanolysin (ALN) or ( b ) listeriolysin O (LLO) or ( c ) the MAC of complement, hCD59 crosslinking (CL-CD59) induces robust RBC necroptosis as indicated by inhibition of RBC death with nec-1 (50 μ M). ALN, LLO, and the MAC all form protein-based pores ≥10 nm in diameter. ( d ) When combined with the hCD59-independent A-tox, which forms protein-based pores of 1–2 nm in diameter, induction of RBC necroptosis by hCD59 is weak. ( e ) When combined with β -hemolysin, which forms polyene-based pores, hCD59 crosslinking fails to induce RBC necroptosis. All PFTs were used at 0.2 HU. ( f ) hCD59 crosslinking does not induce RBC necroptosis when combined with two forms of eryptosis damage, hyperosmotic stress (osm) and excess calcium (cal). All hemolysis assays except for eryptosis were completed at a time point of 30 min. Eryptosis stimuli were added to RBCs over a period of 24 h. When Nec-1 was used, RBCs were treated for 1 h before the addition of stimuli. CL-IgG=crosslinked irrelevant IgG control. Error bars in hemolysis assays represent stand ard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P

    Techniques Used: Inhibition

    Binding or crosslinking of the hCD59 receptor leads to FasL-dependent phosphorylation of RIP1 in RBCs. ( a ) RIP1 IPs showing p-RIP1 in response to binding of hCD59 by the specific mAb MEM-43 (CD59, 1 μ g/mL) or a His-tagged version of the binding domain of VLY (VLYD4, 100 ng/ml). Further crosslinking of hCD59 with an anti-mouse IgG pAb (CL-CD59) or an anti-His mAb (CL-VLYD4) leads to more robust levels of p-RIP1. ( b ) IPs similar to those in ( a ) showing p-RIP1 specifically in response to hCD59 binding. A version of PLY modified with the binding domain of VLY (PLY-VLYD4) induces p-RIP1 while VLY modified with the binding domain of PLY (VLY-PLYD4) does not. RIP1 is phosphorylated specifically in response to CL-CD59 or CL-VLYD4. Crosslinking of hCD55 (CL-CD55) or PLYD4 (CL-PLYD4) does not result in p-RIP1. The p-RIP1 induced by CL-CD59 is retained in combination with ( c ) the hCD59-independent PFTs, PLY or A-tox, or ( d ) the eryptosis stimuli, hyperosmotic stress (osm) or excess calcium (cal). ( e ) The p-RIP1 induced by CL-CD59 or CL-VLYD4 is prevented by the RIP1 inhibitor nec-1 (50 μ M) or neutralization of FasL (NOK-1, 1 μ g/ml). Treatments for all experiments except for eryptosis were 1 h. Eryptosis stimuli were added to RBCs for a period of 24 h. When nec-1 or mAb NOK-1 was used, RBCs were treated with these inhibitors for 1 h before the addition of stimuli
    Figure Legend Snippet: Binding or crosslinking of the hCD59 receptor leads to FasL-dependent phosphorylation of RIP1 in RBCs. ( a ) RIP1 IPs showing p-RIP1 in response to binding of hCD59 by the specific mAb MEM-43 (CD59, 1 μ g/mL) or a His-tagged version of the binding domain of VLY (VLYD4, 100 ng/ml). Further crosslinking of hCD59 with an anti-mouse IgG pAb (CL-CD59) or an anti-His mAb (CL-VLYD4) leads to more robust levels of p-RIP1. ( b ) IPs similar to those in ( a ) showing p-RIP1 specifically in response to hCD59 binding. A version of PLY modified with the binding domain of VLY (PLY-VLYD4) induces p-RIP1 while VLY modified with the binding domain of PLY (VLY-PLYD4) does not. RIP1 is phosphorylated specifically in response to CL-CD59 or CL-VLYD4. Crosslinking of hCD55 (CL-CD55) or PLYD4 (CL-PLYD4) does not result in p-RIP1. The p-RIP1 induced by CL-CD59 is retained in combination with ( c ) the hCD59-independent PFTs, PLY or A-tox, or ( d ) the eryptosis stimuli, hyperosmotic stress (osm) or excess calcium (cal). ( e ) The p-RIP1 induced by CL-CD59 or CL-VLYD4 is prevented by the RIP1 inhibitor nec-1 (50 μ M) or neutralization of FasL (NOK-1, 1 μ g/ml). Treatments for all experiments except for eryptosis were 1 h. Eryptosis stimuli were added to RBCs for a period of 24 h. When nec-1 or mAb NOK-1 was used, RBCs were treated with these inhibitors for 1 h before the addition of stimuli

    Techniques Used: Binding Assay, Modification, Neutralization

    Functional pore formation is necessary for hCD59-induced RBC necroptosis. ( a and b ) Hemolysis assays showing that osmotic protection with dextran (500 000 MW) prevents all RBC death by the RBC necroptosis stimuli VLY and ILY. ( c ) Toxoids of PLY (PdB) and A-tox (A-tox toxoid), which are defective for pore formation, do not induce RBC necroptosis when combined with hCD59 crosslinking (CL-CD59). RBC necroptosis induced by hCD59 crosslinking combined with ( d ) PLY, ( e ) ALN, ( f ) LLO, or ( g ) A-tox is completely prevented by osmoprotection with dextran. Time points for all hemolysis assays were 30 min. PLY, ALN, LLO, and A-tox were used at 0.2 HU. CL-IgG=crosslinked irrelevant IgG control. Error bars in hemolysis assays represent standard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P
    Figure Legend Snippet: Functional pore formation is necessary for hCD59-induced RBC necroptosis. ( a and b ) Hemolysis assays showing that osmotic protection with dextran (500 000 MW) prevents all RBC death by the RBC necroptosis stimuli VLY and ILY. ( c ) Toxoids of PLY (PdB) and A-tox (A-tox toxoid), which are defective for pore formation, do not induce RBC necroptosis when combined with hCD59 crosslinking (CL-CD59). RBC necroptosis induced by hCD59 crosslinking combined with ( d ) PLY, ( e ) ALN, ( f ) LLO, or ( g ) A-tox is completely prevented by osmoprotection with dextran. Time points for all hemolysis assays were 30 min. PLY, ALN, LLO, and A-tox were used at 0.2 HU. CL-IgG=crosslinked irrelevant IgG control. Error bars in hemolysis assays represent standard deviation. All hemolysis assays are the results of three independent experiments. Two-way ANOVA with Bonferroni posttest, *** P

    Techniques Used: Functional Assay, Standard Deviation

    3) Product Images from "CpG-DNA exerts antibacterial effects by protecting immune cells and producing bacteria-reactive antibodies"

    Article Title: CpG-DNA exerts antibacterial effects by protecting immune cells and producing bacteria-reactive antibodies

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34722-y

    Production of bacteria-reactive antibodies in mouse peritoneal cavity cells following treatment with CpG-DNA 1826 in vitro . BALB/c mice were i.p. injected with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. (A) After 7 days, the cells from the peritoneal cavity were harvested and then stimulated with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. After 48 h, the cell culture supernatants were collected, and the amounts of total IgG were determined by ELISA (n = 3/group). (B) The peritoneal cells from PBS-injected mice were stimulated with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. To measure the amounts of antibodies reactive to Gram-positive bacteria, the indicated bacteria were added to poly-L-lysine-coated plates and the cell culture supernatants were applied. The amount of bound IgG was determined by ELISA (n = 3/group). (C) After the i.p. administration of PBS or CpG-DNA 1826 to BALB/c mice, B1 and B2 cells were isolated from the peritoneal cavity using a FACSAria TM II system and fluorescence-labeled anti-mouse CD19 and anti-mouse CD23 antibodies. (D,E) Isolated B1 and B2 cells from the peritoneal cavity were stimulated with PBS or CpG-DNA 1826. After 48 h, the cell culture supernatants were collected and the amount of total IgG (D) and the antibodies reactive to Gram-positive bacteria (E) were determined by ELISA (n = 3/group). 1826, CpG-DNA 1826. 2041, non-CpG-DNA 2041. The results presented are representative of three experiments. ***p
    Figure Legend Snippet: Production of bacteria-reactive antibodies in mouse peritoneal cavity cells following treatment with CpG-DNA 1826 in vitro . BALB/c mice were i.p. injected with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. (A) After 7 days, the cells from the peritoneal cavity were harvested and then stimulated with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. After 48 h, the cell culture supernatants were collected, and the amounts of total IgG were determined by ELISA (n = 3/group). (B) The peritoneal cells from PBS-injected mice were stimulated with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. To measure the amounts of antibodies reactive to Gram-positive bacteria, the indicated bacteria were added to poly-L-lysine-coated plates and the cell culture supernatants were applied. The amount of bound IgG was determined by ELISA (n = 3/group). (C) After the i.p. administration of PBS or CpG-DNA 1826 to BALB/c mice, B1 and B2 cells were isolated from the peritoneal cavity using a FACSAria TM II system and fluorescence-labeled anti-mouse CD19 and anti-mouse CD23 antibodies. (D,E) Isolated B1 and B2 cells from the peritoneal cavity were stimulated with PBS or CpG-DNA 1826. After 48 h, the cell culture supernatants were collected and the amount of total IgG (D) and the antibodies reactive to Gram-positive bacteria (E) were determined by ELISA (n = 3/group). 1826, CpG-DNA 1826. 2041, non-CpG-DNA 2041. The results presented are representative of three experiments. ***p

    Techniques Used: In Vitro, Mouse Assay, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Fluorescence, Labeling

    Production of bacteria-reactive antibodies in the mouse peritoneal cavity and serum following the administration of CpG-DNA 1826. ( A , B ) BALB/c mice were i.p. injected with CpG-DNA 1826. After 7 days, the mice were i.v. injected with S. aureus MW2 (1 × 10 7 CFU). Two days after bacterial infection, the peritoneal fluid and sera were collected from the mice. Bacteria-reactive antibodies in the peritoneal fluid ( A ) and sera ( B ) were captured using S. aureus MW2 coated plates (n = 3/group) and the concentrations of total IgG and each IgG isotype were measured by ELISA. 1826, CpG-DNA 1826. MW2, S. aureus MW2. ( C – F) BALB/c ( C , D ) and TLR9 −/− ( E , F ) mice were i.p. injected with CpG-DNA 1826. Seven days after the administration of CpG-DNA 1826, supernatants were collected from the peritoneal fluid ( C , E ) and sera ( D , F ). To measure the amounts of antibodies reactive to Gram-positive bacteria, the indicated bacteria were added to poly-L-lysine-coated plates and the amount of bound IgG was determined by ELISA (n = 3/group). The results presented are representative of three independent experiments. *p
    Figure Legend Snippet: Production of bacteria-reactive antibodies in the mouse peritoneal cavity and serum following the administration of CpG-DNA 1826. ( A , B ) BALB/c mice were i.p. injected with CpG-DNA 1826. After 7 days, the mice were i.v. injected with S. aureus MW2 (1 × 10 7 CFU). Two days after bacterial infection, the peritoneal fluid and sera were collected from the mice. Bacteria-reactive antibodies in the peritoneal fluid ( A ) and sera ( B ) were captured using S. aureus MW2 coated plates (n = 3/group) and the concentrations of total IgG and each IgG isotype were measured by ELISA. 1826, CpG-DNA 1826. MW2, S. aureus MW2. ( C – F) BALB/c ( C , D ) and TLR9 −/− ( E , F ) mice were i.p. injected with CpG-DNA 1826. Seven days after the administration of CpG-DNA 1826, supernatants were collected from the peritoneal fluid ( C , E ) and sera ( D , F ). To measure the amounts of antibodies reactive to Gram-positive bacteria, the indicated bacteria were added to poly-L-lysine-coated plates and the amount of bound IgG was determined by ELISA (n = 3/group). The results presented are representative of three independent experiments. *p

    Techniques Used: Mouse Assay, Injection, Infection, Enzyme-linked Immunosorbent Assay

    Enhanced phagocytosis induced by the monoclonal antibody produced by CpG-DNA 1826-stimulated mouse peritoneal cavity B cells. ( A ) Production of the bacteria-reactive monoclonal antibody. Ascites from mice induced by the 3F5H6 clone were isolated, and the resulting monoclonal antibody was purified by Protein A affinity column chromatography, subjected to SDS-PAGE, and stained with Coomassie brilliant blue R-250 solution. R, reducing. NR, non-reducing. ( B ) The isotype of the monoclonal antibody was determined by ELISA using S. aureus MW2-coated plates. ( C ) The bacteria-reactivity of the antibody was assessed by ELISA using plates coated with the indicated Gram-positive bacteria (n = 3/group). ( D – G ) FITC-labeled S. aureus MW2 cells (3 × 10 8 CFU/mL) were incubated with PBS, normal mouse IgG, or 3F5H6 mIg (10 μg/mL) for 1 h then added to RAW 264.7 cells ( D,E ) and peritoneal cells ( F,G ) in vitro . After 1 h, the cells were washed with PBS, fixed, and stained with Hoechst No. 33258 to visualize the nuclei (blue). ( D,F ) Confocal images revealed phagocytosis of S. aureus MW2. Scale bars, 10 μm. MW2, S. aureus MW2. ( E,G ) The phagocytic index was analyzed (n = 3/group). ( H,I ) Phagocytosis was enhanced by the bacteria-reactive monoclonal antibody. FITC-labeled S. aureus MW2 cells (3 × 10 8 CFU/mL) were incubated with normal mouse IgG or 3F5H6 mIgG (10 μg/mL) for 1 h and i.p. injected into BALB/c mice. After 1 h, peritoneal cells were harvested from the mice and stained with specific markers for macrophages ( H ) and dendritic cells ( I ). The phagocytic levels were analyzed by flow cytometry (n = 3/group). The results presented are representative of three experiments. *p
    Figure Legend Snippet: Enhanced phagocytosis induced by the monoclonal antibody produced by CpG-DNA 1826-stimulated mouse peritoneal cavity B cells. ( A ) Production of the bacteria-reactive monoclonal antibody. Ascites from mice induced by the 3F5H6 clone were isolated, and the resulting monoclonal antibody was purified by Protein A affinity column chromatography, subjected to SDS-PAGE, and stained with Coomassie brilliant blue R-250 solution. R, reducing. NR, non-reducing. ( B ) The isotype of the monoclonal antibody was determined by ELISA using S. aureus MW2-coated plates. ( C ) The bacteria-reactivity of the antibody was assessed by ELISA using plates coated with the indicated Gram-positive bacteria (n = 3/group). ( D – G ) FITC-labeled S. aureus MW2 cells (3 × 10 8 CFU/mL) were incubated with PBS, normal mouse IgG, or 3F5H6 mIg (10 μg/mL) for 1 h then added to RAW 264.7 cells ( D,E ) and peritoneal cells ( F,G ) in vitro . After 1 h, the cells were washed with PBS, fixed, and stained with Hoechst No. 33258 to visualize the nuclei (blue). ( D,F ) Confocal images revealed phagocytosis of S. aureus MW2. Scale bars, 10 μm. MW2, S. aureus MW2. ( E,G ) The phagocytic index was analyzed (n = 3/group). ( H,I ) Phagocytosis was enhanced by the bacteria-reactive monoclonal antibody. FITC-labeled S. aureus MW2 cells (3 × 10 8 CFU/mL) were incubated with normal mouse IgG or 3F5H6 mIgG (10 μg/mL) for 1 h and i.p. injected into BALB/c mice. After 1 h, peritoneal cells were harvested from the mice and stained with specific markers for macrophages ( H ) and dendritic cells ( I ). The phagocytic levels were analyzed by flow cytometry (n = 3/group). The results presented are representative of three experiments. *p

    Techniques Used: Produced, Mouse Assay, Isolation, Purification, Affinity Column, Chromatography, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Labeling, Incubation, In Vitro, Injection, Flow Cytometry, Cytometry

    4) Product Images from "B Cell-Specific Expression of Ataxia-Telangiectasia Mutated Protein Kinase Promotes Chronic Gammaherpesvirus Infection"

    Article Title: B Cell-Specific Expression of Ataxia-Telangiectasia Mutated Protein Kinase Promotes Chronic Gammaherpesvirus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.01103-17

    ATM deficiency in B cells attenuates MHV68-driven B cell differentiation. B-Cre-positive and -negative mice were either mock treated or infected intranasally with 10 4 PFU of MHV68. At 16 days postinfection (dpi), splenocytes were harvested and analyzed using flow cytometry. Each data point represents an individual mouse; data from 2 to 4 independent experiments were pooled. (A) Class-switched B cells were pregated on B220 + and identified as IgM − IgD − (a representative flow diagram is shown). Boxed areas identify immune populations of interest. (B and C) The frequencies (B) and the absolute numbers (C) of B220 + IgM − IgD − splenocytes were quantified. (D) Germinal center B cells were pregated on B220 + and further identified as CD95 + GL7 + (a representative flow diagram is shown). (E and F) Frequencies (E) and absolute numbers (F) of B220 + CD95 + GL7 + splenocytes. (G) Plasma cells were pregated on B220 + , further gated as IgM − IgD − , and identified for surface expression of CD138 + and for intracellular IgG + (representative flow diagrams are shown). (H and I) Frequencies (H) and absolute numbers (I) of B220 + IgM − IgD − CD138 + IgG + plasma cells. (J to M) Serum total immunoglobulin (J) and MHV68-specific antibodies (total [K], IgM [L], and IgG [M]) were measured by ELISA at 16 days post-mock treatment or -MHV68 infection; pooled data are shown. *, P
    Figure Legend Snippet: ATM deficiency in B cells attenuates MHV68-driven B cell differentiation. B-Cre-positive and -negative mice were either mock treated or infected intranasally with 10 4 PFU of MHV68. At 16 days postinfection (dpi), splenocytes were harvested and analyzed using flow cytometry. Each data point represents an individual mouse; data from 2 to 4 independent experiments were pooled. (A) Class-switched B cells were pregated on B220 + and identified as IgM − IgD − (a representative flow diagram is shown). Boxed areas identify immune populations of interest. (B and C) The frequencies (B) and the absolute numbers (C) of B220 + IgM − IgD − splenocytes were quantified. (D) Germinal center B cells were pregated on B220 + and further identified as CD95 + GL7 + (a representative flow diagram is shown). (E and F) Frequencies (E) and absolute numbers (F) of B220 + CD95 + GL7 + splenocytes. (G) Plasma cells were pregated on B220 + , further gated as IgM − IgD − , and identified for surface expression of CD138 + and for intracellular IgG + (representative flow diagrams are shown). (H and I) Frequencies (H) and absolute numbers (I) of B220 + IgM − IgD − CD138 + IgG + plasma cells. (J to M) Serum total immunoglobulin (J) and MHV68-specific antibodies (total [K], IgM [L], and IgG [M]) were measured by ELISA at 16 days post-mock treatment or -MHV68 infection; pooled data are shown. *, P

    Techniques Used: Cell Differentiation, Mouse Assay, Infection, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Trib1 Is Overexpressed in Systemic Lupus Erythematosus, While It Regulates Immunoglobulin Production in Murine B Cells"

    Article Title: Trib1 Is Overexpressed in Systemic Lupus Erythematosus, While It Regulates Immunoglobulin Production in Murine B Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00373

    Trib1 overexpression in B cells induces a reduction in the production of secreted form of IgG1. Total splenocytes from Trib1-ROSA and Trib1-ROSA Mb1Cre mice were stimulated with LPS/IL-4 for 72 h in vitro [with (+) or without (Ø) the addition of a protein transport inhibitor “GolgiStop™” for the last 8 h of culture], then stained for intracytoplasmic IgG1 after a step of membrane Ig blocking using an anti-murine IgG antibody, a step of fixation and permeabilization, and finally were analyzed by flow cytometry. (A) Percentage of B220 + B cells stained for intracellular IgG1. (B) MFI of intracellular IgG1 staining on B220 + B cells. (C) A representative sample of each condition is shown. Each dot represents the result for one animal.
    Figure Legend Snippet: Trib1 overexpression in B cells induces a reduction in the production of secreted form of IgG1. Total splenocytes from Trib1-ROSA and Trib1-ROSA Mb1Cre mice were stimulated with LPS/IL-4 for 72 h in vitro [with (+) or without (Ø) the addition of a protein transport inhibitor “GolgiStop™” for the last 8 h of culture], then stained for intracytoplasmic IgG1 after a step of membrane Ig blocking using an anti-murine IgG antibody, a step of fixation and permeabilization, and finally were analyzed by flow cytometry. (A) Percentage of B220 + B cells stained for intracellular IgG1. (B) MFI of intracellular IgG1 staining on B220 + B cells. (C) A representative sample of each condition is shown. Each dot represents the result for one animal.

    Techniques Used: Over Expression, Mouse Assay, In Vitro, Staining, Blocking Assay, Flow Cytometry, Cytometry

    6) Product Images from "Vascular Biology and Microcirculation: Potential pitfalls in analyzing structural uncoupling of eNOS: aging is not associated with increased enzyme monomerization"

    Article Title: Vascular Biology and Microcirculation: Potential pitfalls in analyzing structural uncoupling of eNOS: aging is not associated with increased enzyme monomerization

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00506.2018

    Expression of endothelial nitric oxide synthase (eNOS) in lysates from young (Y) and old (O) rat aortas processed under nondenaturing (no β-mercaptoethanol, not boiled) and denaturing (2.5% β-mercaptoethanol, boiled) conditions. Expression of eNOS was assessed by low-temperature SDS-PAGE (4–15%) followed by immunoblot analysis. A–D : representative blots for eNOS, secondary antibody (2ry Ab) only, and Akt, with 4 pairs (nondenatured and denatured) of lysates prepared from 4 different animals. The secondary antibody, a goat anti-mouse IgG (catalog no. 115-035-166, Jackson ImmunoResearch Laboratories), had been purified to minimize cross-reactivity with rat IgG. Representative blots in A and D are the same, with D being overexposed to highlight the weak presence of eNOS monomers in the nondenatured samples. E : combined data for eNOS expression in young and old arteries, relative to Akt expression, comprising the ~220-kDa dimer in nondenaturing conditions ( left ) and the ~135-kDa monomer in denaturing conditions ( right ). Values are means ± SE for 11 young and 10 old aortas from different animals. * indicates a statistically significant difference (** P
    Figure Legend Snippet: Expression of endothelial nitric oxide synthase (eNOS) in lysates from young (Y) and old (O) rat aortas processed under nondenaturing (no β-mercaptoethanol, not boiled) and denaturing (2.5% β-mercaptoethanol, boiled) conditions. Expression of eNOS was assessed by low-temperature SDS-PAGE (4–15%) followed by immunoblot analysis. A–D : representative blots for eNOS, secondary antibody (2ry Ab) only, and Akt, with 4 pairs (nondenatured and denatured) of lysates prepared from 4 different animals. The secondary antibody, a goat anti-mouse IgG (catalog no. 115-035-166, Jackson ImmunoResearch Laboratories), had been purified to minimize cross-reactivity with rat IgG. Representative blots in A and D are the same, with D being overexposed to highlight the weak presence of eNOS monomers in the nondenatured samples. E : combined data for eNOS expression in young and old arteries, relative to Akt expression, comprising the ~220-kDa dimer in nondenaturing conditions ( left ) and the ~135-kDa monomer in denaturing conditions ( right ). Values are means ± SE for 11 young and 10 old aortas from different animals. * indicates a statistically significant difference (** P

    Techniques Used: Expressing, SDS Page, Purification

    Expression of endothelial nitric oxide synthase (eNOS) in lysates from young (Y) and old (O) rat aortas processed under nondenaturing (no β-mercaptoethanol, not boiled) and denaturing (2.5% β-mercaptoethanol, boiled) conditions. Expression of eNOS was assessed by low-temperature SDS-PAGE (4–15%) followed by immunoblot analysis. A–C : representative blots for eNOS, secondary antibody (2ry Ab) only, and Akt, with 4 pairs (nondenatured and denatured) of lysates prepared from 4 different animals. The secondary antibody, a goat anti-mouse IgG (catalog no. 115-036-003, Jackson ImmunoResearch Laboratories), had not been processed to reduce potential cross-reactivity with rat IgG. D : combined data for eNOS expression in young and old arteries, relative to Akt expression, comprising the ~115- to 135-kDa band observed in nondenaturing conditions ( left ), the ~220-kDa dimer in nondenaturing conditions ( middle ), and the ~135-kDa monomer in denaturing conditions ( right ). Values are means ± SE for 11 young and 12 old aortas from different animals. * indicates a statistically significant difference (** P
    Figure Legend Snippet: Expression of endothelial nitric oxide synthase (eNOS) in lysates from young (Y) and old (O) rat aortas processed under nondenaturing (no β-mercaptoethanol, not boiled) and denaturing (2.5% β-mercaptoethanol, boiled) conditions. Expression of eNOS was assessed by low-temperature SDS-PAGE (4–15%) followed by immunoblot analysis. A–C : representative blots for eNOS, secondary antibody (2ry Ab) only, and Akt, with 4 pairs (nondenatured and denatured) of lysates prepared from 4 different animals. The secondary antibody, a goat anti-mouse IgG (catalog no. 115-036-003, Jackson ImmunoResearch Laboratories), had not been processed to reduce potential cross-reactivity with rat IgG. D : combined data for eNOS expression in young and old arteries, relative to Akt expression, comprising the ~115- to 135-kDa band observed in nondenaturing conditions ( left ), the ~220-kDa dimer in nondenaturing conditions ( middle ), and the ~135-kDa monomer in denaturing conditions ( right ). Values are means ± SE for 11 young and 12 old aortas from different animals. * indicates a statistically significant difference (** P

    Techniques Used: Expressing, SDS Page

    A–C : representative blots for expression of endothelial nitric oxide synthase (eNOS) and Akt in lysates of young (Y) and old (O) rat aortas. Lysates were assessed under nondenaturing (not boiled) and denaturing (boiled) conditions in the presence and absence of 2.5% β-mercaptoethanol (β-ME), and protein expression was assessed by low-temperature SDS-PAGE (4–15%) followed by immunoblot analysis. The secondary antibody, a goat anti-mouse IgG (catalog no. 115-035-166, Jackson ImmunoResearch Laboratories), had been purified to minimize cross-reactivity with rat IgG. Representative blots in A and B are the same, with B being overexposed to highlight the weak presence of eNOS oligomers in denatured samples without β-ME.
    Figure Legend Snippet: A–C : representative blots for expression of endothelial nitric oxide synthase (eNOS) and Akt in lysates of young (Y) and old (O) rat aortas. Lysates were assessed under nondenaturing (not boiled) and denaturing (boiled) conditions in the presence and absence of 2.5% β-mercaptoethanol (β-ME), and protein expression was assessed by low-temperature SDS-PAGE (4–15%) followed by immunoblot analysis. The secondary antibody, a goat anti-mouse IgG (catalog no. 115-035-166, Jackson ImmunoResearch Laboratories), had been purified to minimize cross-reactivity with rat IgG. Representative blots in A and B are the same, with B being overexposed to highlight the weak presence of eNOS oligomers in denatured samples without β-ME.

    Techniques Used: Expressing, SDS Page, Purification

    7) Product Images from "Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells"

    Article Title: Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030684

    Kinetics of the p75NTR knockdown effect in PC12 cells. PC12 cells were transiently transfected with SH325-G7-KDEL or the control α phOx-KDEL and harvested at different time points. The p75NTR surface expression levels were determined with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′) 2 fragment APC conjugated (1∶200). The blue histograms represent the p75NTR surface expressions of the PC12 cells expressing SH325-G7-KDEL. The white histograms represent the p75NTR surface expressions of the PC12 cells expressing α phOx-KDEL. The efficiency of knockdown p75NTR surface expression is indicated in percent.
    Figure Legend Snippet: Kinetics of the p75NTR knockdown effect in PC12 cells. PC12 cells were transiently transfected with SH325-G7-KDEL or the control α phOx-KDEL and harvested at different time points. The p75NTR surface expression levels were determined with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′) 2 fragment APC conjugated (1∶200). The blue histograms represent the p75NTR surface expressions of the PC12 cells expressing SH325-G7-KDEL. The white histograms represent the p75NTR surface expressions of the PC12 cells expressing α phOx-KDEL. The efficiency of knockdown p75NTR surface expression is indicated in percent.

    Techniques Used: Transfection, Expressing

    Characteration of the p75NTR-specific antibodies. (A) Antigen binding ELISA. 100 ng of mouse p75NTRex-Fc (shown in dark blue), rat TrkAex-Fc, rat TrkBex-Fc, mouse NgRex-Fc, mouse or human APPex-Fc, N protein SL standard or BSA were immobilized in the plate for each well. 250 ng of each scFv was added after the antigen-coated plates were blocked with FCS for 1.5 hr. Bound scFvs were detected using anti myc-tag mAb (1∶500) and goat anti-mouse IgG HRP conjugated (1∶5,000). (B) The scFvs specifically recognize native p75NTR on PC12 cell surfaces. PC12 or HEK293T cells were stained with 250 ng of the p75NTR-specific scFvs (SH325-A11, SH325-B6, SH325-G7). Bound scFvs were detected by mouse anti-His 6 mAb (1∶100) followed by goat anti-mouse IgG F(ab′) 2 fragment APC conjugated (1∶200). The p75NTR surface expression on PC12 cells was determined by staining PC12 or HEK293T cells with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′) 2 fragment APC conjugated (1∶200). The blue histograms represent the mouse anti-p75NTR mAb (MLR2) or the p75NTR-specific scFvs staining PC12 cells (upper row) and HEK 293T cells (lower row). The white histograms represent the controls stained with goat anti-mouse IgG F(ab′) 2 fragment APC conjugated alone (MLR2 line) or α phOx scFv (other lines) followed by mouse anti-His 6 mAb and goat anti-mouse IgG F(ab′) 2 fragment APC conjugated. ( C ) Detection of denatured antigen (p75NTRex-mFc) by the p75NTR-specific recombinant antibodies in immunoblot. 250 ng of p75NTRex-mFc was denatured and blotted on a PVDF membrane. 1 µg of each p75NTR-specific recombinant antibody was used to stain the membrane for 1.5 hr. The bound antibodies were detected by goat anti-human IgG Fc antiserum AP conjugated (1∶2,000) for 1 hr at RT.
    Figure Legend Snippet: Characteration of the p75NTR-specific antibodies. (A) Antigen binding ELISA. 100 ng of mouse p75NTRex-Fc (shown in dark blue), rat TrkAex-Fc, rat TrkBex-Fc, mouse NgRex-Fc, mouse or human APPex-Fc, N protein SL standard or BSA were immobilized in the plate for each well. 250 ng of each scFv was added after the antigen-coated plates were blocked with FCS for 1.5 hr. Bound scFvs were detected using anti myc-tag mAb (1∶500) and goat anti-mouse IgG HRP conjugated (1∶5,000). (B) The scFvs specifically recognize native p75NTR on PC12 cell surfaces. PC12 or HEK293T cells were stained with 250 ng of the p75NTR-specific scFvs (SH325-A11, SH325-B6, SH325-G7). Bound scFvs were detected by mouse anti-His 6 mAb (1∶100) followed by goat anti-mouse IgG F(ab′) 2 fragment APC conjugated (1∶200). The p75NTR surface expression on PC12 cells was determined by staining PC12 or HEK293T cells with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′) 2 fragment APC conjugated (1∶200). The blue histograms represent the mouse anti-p75NTR mAb (MLR2) or the p75NTR-specific scFvs staining PC12 cells (upper row) and HEK 293T cells (lower row). The white histograms represent the controls stained with goat anti-mouse IgG F(ab′) 2 fragment APC conjugated alone (MLR2 line) or α phOx scFv (other lines) followed by mouse anti-His 6 mAb and goat anti-mouse IgG F(ab′) 2 fragment APC conjugated. ( C ) Detection of denatured antigen (p75NTRex-mFc) by the p75NTR-specific recombinant antibodies in immunoblot. 250 ng of p75NTRex-mFc was denatured and blotted on a PVDF membrane. 1 µg of each p75NTR-specific recombinant antibody was used to stain the membrane for 1.5 hr. The bound antibodies were detected by goat anti-human IgG Fc antiserum AP conjugated (1∶2,000) for 1 hr at RT.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Recombinant

    Knockdown of p75NTR surface expression in PC12 cells by the p75NTR-specific ER retained intrabodies. PC12 cells were transiently transfected with the p75NTR-specific ER retained intrabodies or α phOx-KDEL. Four days after transfection, the p75NTR surface expressions were determined with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′) 2 fragment APC conjugated (1∶200, B, D, F, H, and J). Cells stained with secondary antibody alone were served as control (A, C, E, G, and I). Overlay analysis for the p75NTR surface expression is shown in PC12 cells (K). The blue histograms represent the p75NTR surface expression of the PC12 cells expressing the p75NTR-specific ER retained intrabodies. The white histograms represent the p75NTR surface expression of the PC12 cells expressing the control α phOx-KDEL. The efficiency of knockdown p75NTR surface expression is indicated in percent. The picture of SH325-G7-KDEL is also shown in Figure 7 as 4 day time point result.
    Figure Legend Snippet: Knockdown of p75NTR surface expression in PC12 cells by the p75NTR-specific ER retained intrabodies. PC12 cells were transiently transfected with the p75NTR-specific ER retained intrabodies or α phOx-KDEL. Four days after transfection, the p75NTR surface expressions were determined with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′) 2 fragment APC conjugated (1∶200, B, D, F, H, and J). Cells stained with secondary antibody alone were served as control (A, C, E, G, and I). Overlay analysis for the p75NTR surface expression is shown in PC12 cells (K). The blue histograms represent the p75NTR surface expression of the PC12 cells expressing the p75NTR-specific ER retained intrabodies. The white histograms represent the p75NTR surface expression of the PC12 cells expressing the control α phOx-KDEL. The efficiency of knockdown p75NTR surface expression is indicated in percent. The picture of SH325-G7-KDEL is also shown in Figure 7 as 4 day time point result.

    Techniques Used: Expressing, Transfection, Staining

    Knockdown of p75NTR surface expression in NSC19 cells by the p75NTR-specific ER retained intrabodies. NSC19 cells were transiently transfected with the p75NTR-specific ER retained intrabodies or the control α phOx-KDEL. Four days after transfection, the p75NTR surface expressions were determined with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′) 2 fragment APC conjugated (1∶200). The blue histograms represent the p75NTR surface expressions of the NSC19 cells expressing the p75NTR-specific ER retained intrabodies. The white histograms represent the p75NTR surface expressions of the NSC19 cells expressing the control α phOx-KDEL. The efficiency of knockdown p75NTR surface expression is indicated in percent.
    Figure Legend Snippet: Knockdown of p75NTR surface expression in NSC19 cells by the p75NTR-specific ER retained intrabodies. NSC19 cells were transiently transfected with the p75NTR-specific ER retained intrabodies or the control α phOx-KDEL. Four days after transfection, the p75NTR surface expressions were determined with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′) 2 fragment APC conjugated (1∶200). The blue histograms represent the p75NTR surface expressions of the NSC19 cells expressing the p75NTR-specific ER retained intrabodies. The white histograms represent the p75NTR surface expressions of the NSC19 cells expressing the control α phOx-KDEL. The efficiency of knockdown p75NTR surface expression is indicated in percent.

    Techniques Used: Expressing, Transfection

    8) Product Images from "Trib1 Is Overexpressed in Systemic Lupus Erythematosus, While It Regulates Immunoglobulin Production in Murine B Cells"

    Article Title: Trib1 Is Overexpressed in Systemic Lupus Erythematosus, While It Regulates Immunoglobulin Production in Murine B Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00373

    Trib1 overexpression in B cells induces a reduction in the production of secreted form of IgG1. Total splenocytes from Trib1-ROSA and Trib1-ROSA Mb1Cre mice were stimulated with LPS/IL-4 for 72 h in vitro [with (+) or without (Ø) the addition of a protein transport inhibitor “GolgiStop™” for the last 8 h of culture], then stained for intracytoplasmic IgG1 after a step of membrane Ig blocking using an anti-murine IgG antibody, a step of fixation and permeabilization, and finally were analyzed by flow cytometry. (A) Percentage of B220 + B cells stained for intracellular IgG1. (B) MFI of intracellular IgG1 staining on B220 + B cells. (C) A representative sample of each condition is shown. Each dot represents the result for one animal.
    Figure Legend Snippet: Trib1 overexpression in B cells induces a reduction in the production of secreted form of IgG1. Total splenocytes from Trib1-ROSA and Trib1-ROSA Mb1Cre mice were stimulated with LPS/IL-4 for 72 h in vitro [with (+) or without (Ø) the addition of a protein transport inhibitor “GolgiStop™” for the last 8 h of culture], then stained for intracytoplasmic IgG1 after a step of membrane Ig blocking using an anti-murine IgG antibody, a step of fixation and permeabilization, and finally were analyzed by flow cytometry. (A) Percentage of B220 + B cells stained for intracellular IgG1. (B) MFI of intracellular IgG1 staining on B220 + B cells. (C) A representative sample of each condition is shown. Each dot represents the result for one animal.

    Techniques Used: Over Expression, Mouse Assay, In Vitro, Staining, Blocking Assay, Flow Cytometry, Cytometry

    9) Product Images from "Highly Specific Inhibition of C1q Globular-Head Binding to Human IgG"

    Article Title: Highly Specific Inhibition of C1q Globular-Head Binding to Human IgG

    Journal: Molecular immunology

    doi: 10.1016/j.molimm.2007.12.019

    Western blot analysis of eight CHO-S clones demonstrated various levels of scFv-QuVHVL expression. The blot was probed for 6xHis tagged protein with a mouse anti-6xHis mAb followed by goat anti-mouse IgG-HRP. The target protein was a ∼30kD protein.
    Figure Legend Snippet: Western blot analysis of eight CHO-S clones demonstrated various levels of scFv-QuVHVL expression. The blot was probed for 6xHis tagged protein with a mouse anti-6xHis mAb followed by goat anti-mouse IgG-HRP. The target protein was a ∼30kD protein.

    Techniques Used: Western Blot, Clone Assay, Expressing

    10) Product Images from "The large conductance calcium-activated potassium channel affects extrinsic and intrinsic mechanisms of apoptosis"

    Article Title: The large conductance calcium-activated potassium channel affects extrinsic and intrinsic mechanisms of apoptosis

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.23538

    Results of reciprocal CoIPs following HEK cell transfection with HA-tagged constructs of full-length BK, BK N- or C-terminus only (as marked above each panel), plus either V5-tagged p53 or V5-tagged FADD. A: Results of V5-tagged p53 CoIP experiments ( lanes 1, 4, 7 ) demonstrate that p53 coprecipitates peptide species of ~130, 35, and 100 kDa, respectively. These are the expected weights of full-length BK, BK N-terminus only, and BK C-terminus only. B: The reciprocal experiments show that the three expressed constructs of BK (Full, C- and N-term) precipitate a peptide species of ~56 kDa, the expected weight of p53 ( lanes 1, 4, 7 ). An Lc-specific secondary antibody was used to probe this blot. C: Results of FADD CoIP experiments demonstrate that FADD precipitates peptide species of ~130 and 100 kDa ( lanes 1 and 7) , respectively. These are the expected weights of full-length BK and the C-term of BK. FADD does not precipitate the shorter (~35 kDa) N terminus BK ( lane 4 ). D: The reciprocal experiments show that two expressed constructs of BK (Full and C-term) precipitate a peptide species of ~27 kDa, the expected weight of FADD ( lanes 1, 7 ). Controls are shown in all four panels and consist of IgG-coated beads mixed with lysate in the absence of antibody ( lanes 2, 5, 8 ), and Western blots of lysate alone ( lanes 3, 6, 9 ). Both light (LC) and heavy chain (HC) IgGs are marked appropriately. Designations below each panel designate the primary antibody used to probe the blot.
    Figure Legend Snippet: Results of reciprocal CoIPs following HEK cell transfection with HA-tagged constructs of full-length BK, BK N- or C-terminus only (as marked above each panel), plus either V5-tagged p53 or V5-tagged FADD. A: Results of V5-tagged p53 CoIP experiments ( lanes 1, 4, 7 ) demonstrate that p53 coprecipitates peptide species of ~130, 35, and 100 kDa, respectively. These are the expected weights of full-length BK, BK N-terminus only, and BK C-terminus only. B: The reciprocal experiments show that the three expressed constructs of BK (Full, C- and N-term) precipitate a peptide species of ~56 kDa, the expected weight of p53 ( lanes 1, 4, 7 ). An Lc-specific secondary antibody was used to probe this blot. C: Results of FADD CoIP experiments demonstrate that FADD precipitates peptide species of ~130 and 100 kDa ( lanes 1 and 7) , respectively. These are the expected weights of full-length BK and the C-term of BK. FADD does not precipitate the shorter (~35 kDa) N terminus BK ( lane 4 ). D: The reciprocal experiments show that two expressed constructs of BK (Full and C-term) precipitate a peptide species of ~27 kDa, the expected weight of FADD ( lanes 1, 7 ). Controls are shown in all four panels and consist of IgG-coated beads mixed with lysate in the absence of antibody ( lanes 2, 5, 8 ), and Western blots of lysate alone ( lanes 3, 6, 9 ). Both light (LC) and heavy chain (HC) IgGs are marked appropriately. Designations below each panel designate the primary antibody used to probe the blot.

    Techniques Used: Transfection, Construct, Co-Immunoprecipitation Assay, Western Blot

    11) Product Images from "Proteomic Identification of saeRS-Dependent Targets Critical for Protective Humoral Immunity against Staphylococcus aureus Skin Infection"

    Article Title: Proteomic Identification of saeRS-Dependent Targets Critical for Protective Humoral Immunity against Staphylococcus aureus Skin Infection

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00667-15

    Confirmation of antigen-specific IgG subclass levels by ELISA. (A) There were higher levels of anti-Hla IgG, IgG1, and IgG3 antibodies after S. aureus SSTI in the serum of BALB/c mice than in that of C57BL/6 mice. The higher levels of anti-Hla IgG (B)
    Figure Legend Snippet: Confirmation of antigen-specific IgG subclass levels by ELISA. (A) There were higher levels of anti-Hla IgG, IgG1, and IgG3 antibodies after S. aureus SSTI in the serum of BALB/c mice than in that of C57BL/6 mice. The higher levels of anti-Hla IgG (B)

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

    12) Product Images from "Quantitative Phosphoproteomics Reveals SLP-76 Dependent Regulation of PAG and Src Family Kinases in T Cells"

    Article Title: Quantitative Phosphoproteomics Reveals SLP-76 Dependent Regulation of PAG and Src Family Kinases in T Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046725

    Experimental procedure. Two cell populations of human Jurkat T cell clones (J14-76-11 and J14) were incubated with RPMI 1640 medium containing normal or heavy isotope labeled arginine and lysine amino acids, physically differentiating the two proteomes by a shift in molecular weights. Each cell population was then pre-incubated with OKT3 and OKT4 antibodies for 10 minutes at 4°C and then crosslinked with anti-IgG at 37°C for the times indicated. After cell lysis, light and heavy cell lysates were combined at an equal protein concentration ratio for each timepoint. Proteins were then reduced, alkylated, and trypsin-digested into peptides. Peptides were desalted by Sep-Pak cartridges, enriched by phosphotyrosine peptide immunoprecipitation and Fe 3+ IMAC, and then subjected to reversed-phase LC-MS/MS analysis. MS shifts introduced by heavy isotope labeling allow for differentiation between light and heavy peptide counterparts in MS spectra. Selected ion chromatogram (SIC) peak areas of light and heavy isotope labeled phosphopeptides were calculated for relative quantification of peptide abundance. Individual SIC peak areas were normalized to the SIC peak area of the copurified synthetic peptide LIEDAEpYTAK in the same timepoint. A label-free heatmap was generated based on peptide abundance for a certain peptide in SLP-76 reconstituted Jurkat cells through a time course of receptor stimulation and SILAC ratio heatmaps were generated based on the ratio of abundance between light (SLP-76 reconstituted) and heavy (SLP-76 deficient) peptide counterparts for each timepoint (SLP-76 deficient in relative to SLP-76 reconstituted).
    Figure Legend Snippet: Experimental procedure. Two cell populations of human Jurkat T cell clones (J14-76-11 and J14) were incubated with RPMI 1640 medium containing normal or heavy isotope labeled arginine and lysine amino acids, physically differentiating the two proteomes by a shift in molecular weights. Each cell population was then pre-incubated with OKT3 and OKT4 antibodies for 10 minutes at 4°C and then crosslinked with anti-IgG at 37°C for the times indicated. After cell lysis, light and heavy cell lysates were combined at an equal protein concentration ratio for each timepoint. Proteins were then reduced, alkylated, and trypsin-digested into peptides. Peptides were desalted by Sep-Pak cartridges, enriched by phosphotyrosine peptide immunoprecipitation and Fe 3+ IMAC, and then subjected to reversed-phase LC-MS/MS analysis. MS shifts introduced by heavy isotope labeling allow for differentiation between light and heavy peptide counterparts in MS spectra. Selected ion chromatogram (SIC) peak areas of light and heavy isotope labeled phosphopeptides were calculated for relative quantification of peptide abundance. Individual SIC peak areas were normalized to the SIC peak area of the copurified synthetic peptide LIEDAEpYTAK in the same timepoint. A label-free heatmap was generated based on peptide abundance for a certain peptide in SLP-76 reconstituted Jurkat cells through a time course of receptor stimulation and SILAC ratio heatmaps were generated based on the ratio of abundance between light (SLP-76 reconstituted) and heavy (SLP-76 deficient) peptide counterparts for each timepoint (SLP-76 deficient in relative to SLP-76 reconstituted).

    Techniques Used: Clone Assay, Incubation, Labeling, Lysis, Protein Concentration, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Generated

    13) Product Images from "Masking of Phosphatidylserine Inhibits Apoptotic Cell Engulfment and Induces Autoantibody Production in Mice"

    Article Title: Masking of Phosphatidylserine Inhibits Apoptotic Cell Engulfment and Induces Autoantibody Production in Mice

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20040342

    Immunohistochemical detection of IgG deposition in the glomeruli of kidneys in mice injected with D89E. C57BL/6 mice were injected intravenously with D89E or E1E2PT with or without apoptotic thymocytes six times at weekly intervals. Kidneys obtained 17 wk after the initial injection were fixed with 4% paraformaldehyde and embedded in paraffin. The sections were stained with Cy3-conjugated F(ab′)2 of goat anti-mouse IgG and were observed with fluorescence microscopy. 25 wk-old MRL/lpr mice (E) were used as a positive control. (A) 0.5 μg/ml D89E, (B) 2.0 μg/ml E1E2PT, (C) 2.0 × 10 7 UV-irradiated thymocytes, and (D) 2.0 μg/ml D89E with 2.0 × 10 7 of UV-irradiated thymocytes.
    Figure Legend Snippet: Immunohistochemical detection of IgG deposition in the glomeruli of kidneys in mice injected with D89E. C57BL/6 mice were injected intravenously with D89E or E1E2PT with or without apoptotic thymocytes six times at weekly intervals. Kidneys obtained 17 wk after the initial injection were fixed with 4% paraformaldehyde and embedded in paraffin. The sections were stained with Cy3-conjugated F(ab′)2 of goat anti-mouse IgG and were observed with fluorescence microscopy. 25 wk-old MRL/lpr mice (E) were used as a positive control. (A) 0.5 μg/ml D89E, (B) 2.0 μg/ml E1E2PT, (C) 2.0 × 10 7 UV-irradiated thymocytes, and (D) 2.0 μg/ml D89E with 2.0 × 10 7 of UV-irradiated thymocytes.

    Techniques Used: Immunohistochemistry, Mouse Assay, Injection, Staining, Fluorescence, Microscopy, Positive Control, Irradiation

    14) Product Images from "CpG-DNA exerts antibacterial effects by protecting immune cells and producing bacteria-reactive antibodies"

    Article Title: CpG-DNA exerts antibacterial effects by protecting immune cells and producing bacteria-reactive antibodies

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34722-y

    Production of bacteria-reactive antibodies in mouse peritoneal cavity cells following treatment with CpG-DNA 1826 in vitro . BALB/c mice were i.p. injected with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. (A) After 7 days, the cells from the peritoneal cavity were harvested and then stimulated with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. After 48 h, the cell culture supernatants were collected, and the amounts of total IgG were determined by ELISA (n = 3/group). (B) The peritoneal cells from PBS-injected mice were stimulated with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. To measure the amounts of antibodies reactive to Gram-positive bacteria, the indicated bacteria were added to poly-L-lysine-coated plates and the cell culture supernatants were applied. The amount of bound IgG was determined by ELISA (n = 3/group). (C) After the i.p. administration of PBS or CpG-DNA 1826 to BALB/c mice, B1 and B2 cells were isolated from the peritoneal cavity using a FACSAria TM II system and fluorescence-labeled anti-mouse CD19 and anti-mouse CD23 antibodies. (D,E) Isolated B1 and B2 cells from the peritoneal cavity were stimulated with PBS or CpG-DNA 1826. After 48 h, the cell culture supernatants were collected and the amount of total IgG (D) and the antibodies reactive to Gram-positive bacteria (E) were determined by ELISA (n = 3/group). 1826, CpG-DNA 1826. 2041, non-CpG-DNA 2041. The results presented are representative of three experiments. ***p
    Figure Legend Snippet: Production of bacteria-reactive antibodies in mouse peritoneal cavity cells following treatment with CpG-DNA 1826 in vitro . BALB/c mice were i.p. injected with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. (A) After 7 days, the cells from the peritoneal cavity were harvested and then stimulated with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. After 48 h, the cell culture supernatants were collected, and the amounts of total IgG were determined by ELISA (n = 3/group). (B) The peritoneal cells from PBS-injected mice were stimulated with PBS, CpG-DNA 1826, or non-CpG-DNA 2041. To measure the amounts of antibodies reactive to Gram-positive bacteria, the indicated bacteria were added to poly-L-lysine-coated plates and the cell culture supernatants were applied. The amount of bound IgG was determined by ELISA (n = 3/group). (C) After the i.p. administration of PBS or CpG-DNA 1826 to BALB/c mice, B1 and B2 cells were isolated from the peritoneal cavity using a FACSAria TM II system and fluorescence-labeled anti-mouse CD19 and anti-mouse CD23 antibodies. (D,E) Isolated B1 and B2 cells from the peritoneal cavity were stimulated with PBS or CpG-DNA 1826. After 48 h, the cell culture supernatants were collected and the amount of total IgG (D) and the antibodies reactive to Gram-positive bacteria (E) were determined by ELISA (n = 3/group). 1826, CpG-DNA 1826. 2041, non-CpG-DNA 2041. The results presented are representative of three experiments. ***p

    Techniques Used: In Vitro, Mouse Assay, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Fluorescence, Labeling

    Production of bacteria-reactive antibodies in the mouse peritoneal cavity and serum following the administration of CpG-DNA 1826. ( A , B ) BALB/c mice were i.p. injected with CpG-DNA 1826. After 7 days, the mice were i.v. injected with S. aureus MW2 (1 × 10 7 CFU). Two days after bacterial infection, the peritoneal fluid and sera were collected from the mice. Bacteria-reactive antibodies in the peritoneal fluid ( A ) and sera ( B ) were captured using S. aureus MW2 coated plates (n = 3/group) and the concentrations of total IgG and each IgG isotype were measured by ELISA. 1826, CpG-DNA 1826. MW2, S. aureus MW2. ( C – F) BALB/c ( C , D ) and TLR9 −/− ( E , F ) mice were i.p. injected with CpG-DNA 1826. Seven days after the administration of CpG-DNA 1826, supernatants were collected from the peritoneal fluid ( C , E ) and sera ( D , F ). To measure the amounts of antibodies reactive to Gram-positive bacteria, the indicated bacteria were added to poly-L-lysine-coated plates and the amount of bound IgG was determined by ELISA (n = 3/group). The results presented are representative of three independent experiments. *p
    Figure Legend Snippet: Production of bacteria-reactive antibodies in the mouse peritoneal cavity and serum following the administration of CpG-DNA 1826. ( A , B ) BALB/c mice were i.p. injected with CpG-DNA 1826. After 7 days, the mice were i.v. injected with S. aureus MW2 (1 × 10 7 CFU). Two days after bacterial infection, the peritoneal fluid and sera were collected from the mice. Bacteria-reactive antibodies in the peritoneal fluid ( A ) and sera ( B ) were captured using S. aureus MW2 coated plates (n = 3/group) and the concentrations of total IgG and each IgG isotype were measured by ELISA. 1826, CpG-DNA 1826. MW2, S. aureus MW2. ( C – F) BALB/c ( C , D ) and TLR9 −/− ( E , F ) mice were i.p. injected with CpG-DNA 1826. Seven days after the administration of CpG-DNA 1826, supernatants were collected from the peritoneal fluid ( C , E ) and sera ( D , F ). To measure the amounts of antibodies reactive to Gram-positive bacteria, the indicated bacteria were added to poly-L-lysine-coated plates and the amount of bound IgG was determined by ELISA (n = 3/group). The results presented are representative of three independent experiments. *p

    Techniques Used: Mouse Assay, Injection, Infection, Enzyme-linked Immunosorbent Assay

    Enhanced phagocytosis induced by the monoclonal antibody produced by CpG-DNA 1826-stimulated mouse peritoneal cavity B cells. ( A ) Production of the bacteria-reactive monoclonal antibody. Ascites from mice induced by the 3F5H6 clone were isolated, and the resulting monoclonal antibody was purified by Protein A affinity column chromatography, subjected to SDS-PAGE, and stained with Coomassie brilliant blue R-250 solution. R, reducing. NR, non-reducing. ( B ) The isotype of the monoclonal antibody was determined by ELISA using S. aureus MW2-coated plates. ( C ) The bacteria-reactivity of the antibody was assessed by ELISA using plates coated with the indicated Gram-positive bacteria (n = 3/group). ( D – G ) FITC-labeled S. aureus MW2 cells (3 × 10 8 CFU/mL) were incubated with PBS, normal mouse IgG, or 3F5H6 mIg (10 μg/mL) for 1 h then added to RAW 264.7 cells ( D,E ) and peritoneal cells ( F,G ) in vitro . After 1 h, the cells were washed with PBS, fixed, and stained with Hoechst No. 33258 to visualize the nuclei (blue). ( D,F ) Confocal images revealed phagocytosis of S. aureus MW2. Scale bars, 10 μm. MW2, S. aureus MW2. ( E,G ) The phagocytic index was analyzed (n = 3/group). ( H,I ) Phagocytosis was enhanced by the bacteria-reactive monoclonal antibody. FITC-labeled S. aureus MW2 cells (3 × 10 8 CFU/mL) were incubated with normal mouse IgG or 3F5H6 mIgG (10 μg/mL) for 1 h and i.p. injected into BALB/c mice. After 1 h, peritoneal cells were harvested from the mice and stained with specific markers for macrophages ( H ) and dendritic cells ( I ). The phagocytic levels were analyzed by flow cytometry (n = 3/group). The results presented are representative of three experiments. *p
    Figure Legend Snippet: Enhanced phagocytosis induced by the monoclonal antibody produced by CpG-DNA 1826-stimulated mouse peritoneal cavity B cells. ( A ) Production of the bacteria-reactive monoclonal antibody. Ascites from mice induced by the 3F5H6 clone were isolated, and the resulting monoclonal antibody was purified by Protein A affinity column chromatography, subjected to SDS-PAGE, and stained with Coomassie brilliant blue R-250 solution. R, reducing. NR, non-reducing. ( B ) The isotype of the monoclonal antibody was determined by ELISA using S. aureus MW2-coated plates. ( C ) The bacteria-reactivity of the antibody was assessed by ELISA using plates coated with the indicated Gram-positive bacteria (n = 3/group). ( D – G ) FITC-labeled S. aureus MW2 cells (3 × 10 8 CFU/mL) were incubated with PBS, normal mouse IgG, or 3F5H6 mIg (10 μg/mL) for 1 h then added to RAW 264.7 cells ( D,E ) and peritoneal cells ( F,G ) in vitro . After 1 h, the cells were washed with PBS, fixed, and stained with Hoechst No. 33258 to visualize the nuclei (blue). ( D,F ) Confocal images revealed phagocytosis of S. aureus MW2. Scale bars, 10 μm. MW2, S. aureus MW2. ( E,G ) The phagocytic index was analyzed (n = 3/group). ( H,I ) Phagocytosis was enhanced by the bacteria-reactive monoclonal antibody. FITC-labeled S. aureus MW2 cells (3 × 10 8 CFU/mL) were incubated with normal mouse IgG or 3F5H6 mIgG (10 μg/mL) for 1 h and i.p. injected into BALB/c mice. After 1 h, peritoneal cells were harvested from the mice and stained with specific markers for macrophages ( H ) and dendritic cells ( I ). The phagocytic levels were analyzed by flow cytometry (n = 3/group). The results presented are representative of three experiments. *p

    Techniques Used: Produced, Mouse Assay, Isolation, Purification, Affinity Column, Chromatography, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Labeling, Incubation, In Vitro, Injection, Flow Cytometry, Cytometry

    15) Product Images from "Small, Membrane-bound, Alternatively Spliced Forms of Ankyrin 1 Associated with the Sarcoplasmic Reticulum of Mammalian Skeletal Muscle"

    Article Title: Small, Membrane-bound, Alternatively Spliced Forms of Ankyrin 1 Associated with the Sarcoplasmic Reticulum of Mammalian Skeletal Muscle

    Journal: The Journal of Cell Biology

    doi:

    Comparison of the distribution of the small ankyrins and the SERCA1 ATPase in rat skeletal muscle. Frozen cross sections (5 μm) of the extensor digitorum longus muscle of the rat were prepared and double-labeled by immunofluorescence with anti-p6 antibodies to the small ankyrins and monoclonal antibodies to the SERCA1 ATPase of fast twitch muscle fibers, as described in Materials and Methods. Images were obtained by confocal microscopy. ( A ) SERCA ATPase, visualized with fluoresceinated anti-mouse IgG. ( B ) Small ankyrins, visualized with tetramethylrhodamine-conjugated anti-rabbit IgG. ( C ) Computer overlay of the images in A and B , in which structures labeled by both antibodies appear yellow. There is extensive coincidence of the two labels. Bar, 6 μm.
    Figure Legend Snippet: Comparison of the distribution of the small ankyrins and the SERCA1 ATPase in rat skeletal muscle. Frozen cross sections (5 μm) of the extensor digitorum longus muscle of the rat were prepared and double-labeled by immunofluorescence with anti-p6 antibodies to the small ankyrins and monoclonal antibodies to the SERCA1 ATPase of fast twitch muscle fibers, as described in Materials and Methods. Images were obtained by confocal microscopy. ( A ) SERCA ATPase, visualized with fluoresceinated anti-mouse IgG. ( B ) Small ankyrins, visualized with tetramethylrhodamine-conjugated anti-rabbit IgG. ( C ) Computer overlay of the images in A and B , in which structures labeled by both antibodies appear yellow. There is extensive coincidence of the two labels. Bar, 6 μm.

    Techniques Used: Labeling, Immunofluorescence, Confocal Microscopy

    16) Product Images from "A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis"

    Article Title: A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20052349

    BLT1 is required for synovial inflammatory chemokine and cytokine production in inflammatory arthritis. (a) K/BxN serum was administered to WT and BLT1 −/− mice, and ankles were harvested at 7 d. Frozen ankle sections were stained with FITC-labeled F(ab′) 2 fragment directed against the Fc portion of mouse IgG. A WT mouse that did not receive any serum was used as a control. Bar, 230 μm. (b–e) Chemokine expression was measured by qPCR of total RNA isolated from synovial tissue on postserum transfer days 3 (early onset disease) (b) or 7 (early active disease) (c). Chemokine receptor expression was quantified by qPCR of total RNA isolated from synovial fluid at days 3 (d) and 7 (e). Total RNA was extracted from eight individual ankles in each experimental group, and qPCR reactions were run separately on each sample. Error bars represent SEM. *, P
    Figure Legend Snippet: BLT1 is required for synovial inflammatory chemokine and cytokine production in inflammatory arthritis. (a) K/BxN serum was administered to WT and BLT1 −/− mice, and ankles were harvested at 7 d. Frozen ankle sections were stained with FITC-labeled F(ab′) 2 fragment directed against the Fc portion of mouse IgG. A WT mouse that did not receive any serum was used as a control. Bar, 230 μm. (b–e) Chemokine expression was measured by qPCR of total RNA isolated from synovial tissue on postserum transfer days 3 (early onset disease) (b) or 7 (early active disease) (c). Chemokine receptor expression was quantified by qPCR of total RNA isolated from synovial fluid at days 3 (d) and 7 (e). Total RNA was extracted from eight individual ankles in each experimental group, and qPCR reactions were run separately on each sample. Error bars represent SEM. *, P

    Techniques Used: Mouse Assay, Staining, Labeling, Expressing, Real-time Polymerase Chain Reaction, Isolation

    17) Product Images from "An agonist antibody that blocks autoimmunity by inducing anti-inflammatory macrophages"

    Article Title: An agonist antibody that blocks autoimmunity by inducing anti-inflammatory macrophages

    Journal: The FASEB Journal

    doi: 10.1096/fj.15-281329

    Treatment with LKAb reduces lupus-like disease in MRL-lpr mice. Mice (6–7/group) were injected intraperitoneally with LKAb or PBS (from the age of 6 wk until termination of the experiment at 20 wk) and followed for manifestations of disease . A ) IgG2a anti-chromatin autoantibody levels determined by ELISA at 12 wk of age . B ) Progression of lymphadenopathy between 12 and 16 wk of age assessed by palpation of axillary and salivary lymph nodes (LNs) and scored on a 0–4 scale . C ) Kidney disease determined by histologic examination for GN at 20 wk of age. Representative images for treated and control mice are shown . D ) Kaplan-Meier plot representing survival rates of treated and control mice. * P
    Figure Legend Snippet: Treatment with LKAb reduces lupus-like disease in MRL-lpr mice. Mice (6–7/group) were injected intraperitoneally with LKAb or PBS (from the age of 6 wk until termination of the experiment at 20 wk) and followed for manifestations of disease . A ) IgG2a anti-chromatin autoantibody levels determined by ELISA at 12 wk of age . B ) Progression of lymphadenopathy between 12 and 16 wk of age assessed by palpation of axillary and salivary lymph nodes (LNs) and scored on a 0–4 scale . C ) Kidney disease determined by histologic examination for GN at 20 wk of age. Representative images for treated and control mice are shown . D ) Kaplan-Meier plot representing survival rates of treated and control mice. * P

    Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    18) Product Images from "Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2( SK2) in mouse cardiac myocytes, et al. Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes"

    Article Title: Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2( SK2) in mouse cardiac myocytes, et al. Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes

    Journal: Acta Physiologica (Oxford, England)

    doi: 10.1111/apha.12986

    Colocalizations of JP 2 and SK 2 channels in adult mouse cardiac myocytes and HEK 293 cells. Confocal images show double immunostaining with anti‐ JP 2 antibody (green) and anti‐ SK 2 antibody (red) in single isolated mouse atria (A) and ventricular (B) myocytes as well as HEK 293 cells (D) cotransfected with JP 2 and SK 2. Negative control experiments (Control) were performed with the secondary antibodies with anti‐rabbit‐IgG TRITC ‐conjugated and with anti‐mouse‐IgG FITC ‐conjugated in atria myocytes (C) and HEK 293 cells (E). Merge images show the colocalization of JP 2 and SK 2 channels near the Z ‐lines in the cardiac cells or on the surface membrane of HEK 293 cells. Scale bars, 10 μm
    Figure Legend Snippet: Colocalizations of JP 2 and SK 2 channels in adult mouse cardiac myocytes and HEK 293 cells. Confocal images show double immunostaining with anti‐ JP 2 antibody (green) and anti‐ SK 2 antibody (red) in single isolated mouse atria (A) and ventricular (B) myocytes as well as HEK 293 cells (D) cotransfected with JP 2 and SK 2. Negative control experiments (Control) were performed with the secondary antibodies with anti‐rabbit‐IgG TRITC ‐conjugated and with anti‐mouse‐IgG FITC ‐conjugated in atria myocytes (C) and HEK 293 cells (E). Merge images show the colocalization of JP 2 and SK 2 channels near the Z ‐lines in the cardiac cells or on the surface membrane of HEK 293 cells. Scale bars, 10 μm

    Techniques Used: Double Immunostaining, Isolation, Negative Control

    19) Product Images from "Dendritic cell targeted HIV‐1 gag protein vaccine provides help to a recombinant Newcastle disease virus vectored vaccine including mobilization of protective CD8+ T cells"

    Article Title: Dendritic cell targeted HIV‐1 gag protein vaccine provides help to a recombinant Newcastle disease virus vectored vaccine including mobilization of protective CD8+ T cells

    Journal: Immunity, Inflammation and Disease

    doi: 10.1002/iid3.209

    CD4 + and CD8 + T cells protect mice after DEC‐gag protein‐prime rNDV‐L‐gag‐boost vaccine. Mice vaccinated as indicated in x‐axis of Figure 3 (a–d) above were treated as indicated with control rat IgG or depleting antibodies to αCD4, αCD8, or both (αCD4 αCD8) at days −3, −2, −1 prior to airway challenge with recombinant vaccinia‐gag virus. Lung virus titer (PFU/lung) was determined 7 days after challenge with recombinant vaccinia‐gag virus (Mean ± SD of three repeat experiments) (ns, not significant (two tailed student's t test).
    Figure Legend Snippet: CD4 + and CD8 + T cells protect mice after DEC‐gag protein‐prime rNDV‐L‐gag‐boost vaccine. Mice vaccinated as indicated in x‐axis of Figure 3 (a–d) above were treated as indicated with control rat IgG or depleting antibodies to αCD4, αCD8, or both (αCD4 αCD8) at days −3, −2, −1 prior to airway challenge with recombinant vaccinia‐gag virus. Lung virus titer (PFU/lung) was determined 7 days after challenge with recombinant vaccinia‐gag virus (Mean ± SD of three repeat experiments) (ns, not significant (two tailed student's t test).

    Techniques Used: Mouse Assay, Recombinant, Two Tailed Test

    20) Product Images from "Therapeutic evaluation of monoclonal antibody-maytansinoid conjugate as a model of RON-targeted drug delivery for pancreatic cancer treatment"

    Article Title: Therapeutic evaluation of monoclonal antibody-maytansinoid conjugate as a model of RON-targeted drug delivery for pancreatic cancer treatment

    Journal: American Journal of Cancer Research

    doi:

    Induction of RON endocytosis by Zt/g4-DM1 in PDAC cell lines: (A) Levels of RON expression by PDAC cell lines. Four PDAC cell lines (1 × 10 6 cells/ml) in PBS were incubated at 4°C with 5 μg/ml Zt/g4 for 60 min. Isotope matched mouse IgG was used as the control. Cell surface RON was quantitatively determined by immunofluorescence analysis using QIFKIT® (DAKO). (B) Kinetic reduction of cell surface RON. BxPC-3, FG and L3.6pl cells (1 × 10 6 ], and then incubated with 2 μg/mL of anti-RON mAb Zt/F2. Immunofluorescence was analyzed by flow cytometer using FITC-coupled anti-mouse IgG. Immunofluorescence from cells treated with Zt/g4-DM1 at 4°C was set as 100%. Internalization efficiency (IC 50 ) was calculated as the time required to achieve the 50% reduction of cell surface RON. (C) Immunofluorescent localization of endocytic RON in cytoplasm. BxPC-3, FG, and L3.6pl cells (1 × 10 5 cells per chamber) were treated at 4°C or 37°C with 5 μg/ml of Zt/g4-DM1 for 12 h followed by FITC-coupled goat anti-mouse IgG. LAMP1 was detected using mouse anti-LAMP1 mAb and used as a marker for protein cytoplasmic localization. After cell fixation, immunofluorescence was detected using the BK70 Olympus microscope equipped with a fluorescence apparatus. DAPI was used to stain nuclear DNA. Images were also overlapped to show the co-localization of RON with LAMP1 in cytoplasm.
    Figure Legend Snippet: Induction of RON endocytosis by Zt/g4-DM1 in PDAC cell lines: (A) Levels of RON expression by PDAC cell lines. Four PDAC cell lines (1 × 10 6 cells/ml) in PBS were incubated at 4°C with 5 μg/ml Zt/g4 for 60 min. Isotope matched mouse IgG was used as the control. Cell surface RON was quantitatively determined by immunofluorescence analysis using QIFKIT® (DAKO). (B) Kinetic reduction of cell surface RON. BxPC-3, FG and L3.6pl cells (1 × 10 6 ], and then incubated with 2 μg/mL of anti-RON mAb Zt/F2. Immunofluorescence was analyzed by flow cytometer using FITC-coupled anti-mouse IgG. Immunofluorescence from cells treated with Zt/g4-DM1 at 4°C was set as 100%. Internalization efficiency (IC 50 ) was calculated as the time required to achieve the 50% reduction of cell surface RON. (C) Immunofluorescent localization of endocytic RON in cytoplasm. BxPC-3, FG, and L3.6pl cells (1 × 10 5 cells per chamber) were treated at 4°C or 37°C with 5 μg/ml of Zt/g4-DM1 for 12 h followed by FITC-coupled goat anti-mouse IgG. LAMP1 was detected using mouse anti-LAMP1 mAb and used as a marker for protein cytoplasmic localization. After cell fixation, immunofluorescence was detected using the BK70 Olympus microscope equipped with a fluorescence apparatus. DAPI was used to stain nuclear DNA. Images were also overlapped to show the co-localization of RON with LAMP1 in cytoplasm.

    Techniques Used: Expressing, Incubation, Immunofluorescence, Flow Cytometry, Cytometry, Marker, Microscopy, Fluorescence, Staining

    21) Product Images from "Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas"

    Article Title: Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2018.08.002

    Humanized IL-13Rα2-Targeting CAR T Cells (A) Flow cytometric detection of CAR expression by human T cells, after mRNA electroporation of murine and humanized scFv- (07 and 08) based CAR constructs using rabbit anti-mouse or rabbit anti-human IgG antibodies. (B) Vector maps of tested anti-IL-13Rα2 CAR design based on the size of each components. (C) CAR expression staining of the humanized IL-13Rα2 CAR transduced T cells used in the co-culture experiments. (D) IL-13Rα1 and IL-13Rα2 expression analysis on the human tumor cell lines (Sup-T1, Jurkat, A549, U87, U251, and D270) with isotype antibodies staining control in blue. (E) Flow-based intracellular cytokine (IFNγ) staining of the humanized IL-13Rα2 CAR T cells co-cultured with human tumor cell lines in (D) controlled with un-transduced T cells (UTD). Human CD8 was stained to distinguish the CD4-positive and CD8-positive subgroups of T cells along the x axis. (F) Chromium release assays of humanized IL-13Rα2 CAR T cells co-cultured with tumor cell lines in (D) was analyzed at different effector/target (E:T) ratios (1:1, 3:1, 10:1, and 30:1) compared with the UTD T cells with one-way ANOVA post hoc Tukey test. **p
    Figure Legend Snippet: Humanized IL-13Rα2-Targeting CAR T Cells (A) Flow cytometric detection of CAR expression by human T cells, after mRNA electroporation of murine and humanized scFv- (07 and 08) based CAR constructs using rabbit anti-mouse or rabbit anti-human IgG antibodies. (B) Vector maps of tested anti-IL-13Rα2 CAR design based on the size of each components. (C) CAR expression staining of the humanized IL-13Rα2 CAR transduced T cells used in the co-culture experiments. (D) IL-13Rα1 and IL-13Rα2 expression analysis on the human tumor cell lines (Sup-T1, Jurkat, A549, U87, U251, and D270) with isotype antibodies staining control in blue. (E) Flow-based intracellular cytokine (IFNγ) staining of the humanized IL-13Rα2 CAR T cells co-cultured with human tumor cell lines in (D) controlled with un-transduced T cells (UTD). Human CD8 was stained to distinguish the CD4-positive and CD8-positive subgroups of T cells along the x axis. (F) Chromium release assays of humanized IL-13Rα2 CAR T cells co-cultured with tumor cell lines in (D) was analyzed at different effector/target (E:T) ratios (1:1, 3:1, 10:1, and 30:1) compared with the UTD T cells with one-way ANOVA post hoc Tukey test. **p

    Techniques Used: Flow Cytometry, Expressing, Electroporation, Construct, Plasmid Preparation, Staining, Co-Culture Assay, Cell Culture

    22) Product Images from "Calcineurin-GATA-6 pathway is involved in smooth muscle-specific transcription"

    Article Title: Calcineurin-GATA-6 pathway is involved in smooth muscle-specific transcription

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200106057

    CysA and FK506 repressed the endogenous expression of the Sm-MHC in differentiated VSMCs. VSMCs were cultured in proliferation medium (A) or in differentiation medium in the presence of CysA (D, 0.5 μg/ml), FK506 (E, 1 nM) or nifedipine (F, 1 μM), or in their absence (B and C). These cells were subjected to immunostaining with the anti–Sm-MHC antibody. The primary antibody was further incubated with a secondary antibody conjugated with peroxidase (brown signals). The primary antibody was replaced with normal mouse IgG in C. The nuclei were counterstained with hematoxylin. Bar, 20 μm.
    Figure Legend Snippet: CysA and FK506 repressed the endogenous expression of the Sm-MHC in differentiated VSMCs. VSMCs were cultured in proliferation medium (A) or in differentiation medium in the presence of CysA (D, 0.5 μg/ml), FK506 (E, 1 nM) or nifedipine (F, 1 μM), or in their absence (B and C). These cells were subjected to immunostaining with the anti–Sm-MHC antibody. The primary antibody was further incubated with a secondary antibody conjugated with peroxidase (brown signals). The primary antibody was replaced with normal mouse IgG in C. The nuclei were counterstained with hematoxylin. Bar, 20 μm.

    Techniques Used: Expressing, Cell Culture, Immunostaining, Incubation

    The amount of GATA-6-DNA binding is up-regulated by induction of the differentiated phenotype. (A) Nuclear extracts were obtained from VSMCs with proliferative (P) or differentiated (D) phenotype. These extracts were probed with a radiolabeled oligonucleotide containing the Sm-MHC GATA site. Unlabeled competitor DNAs were present at a 100-fold molar excess as indicated: (lane 4) wild-type Sm-MHC GATA (wt); (lane 5) Sm-MHC GATA with a mutation (mut). The arrow indicates the complex corresponding to the GATA-specific interaction between the Sm-MHC GATA site and GATA-6. B, EMSA studies using the Sm-MHC-GATA probe were performed in nuclear extracts from VSMCs with differentiated (D) phenotype. Equal amounts of the anti–GATA-6 antibody (lane 3), the anti–GATA-4 antibody (lane 2), or normal rabbit IgG (lane 1) were added to the binding mixture. (C) EMSA studies using the probe for Sp-1 were performed in nuclear extracts from VSMCs with proliferative (P) or differentiated (D) phenotype as indicated. The arrow indicates the complex corresponding to the interaction between the probe and Sp-1. (D) The amount of GATA-6-DNA binding in A (lanes 1–3) and that of SP-1-DNA binding in C was quantified by densitometry using NIH image 1.61 and the relative DNA binding amount (Sm-MHC GATA/SP-1) was determined. The relative DNA binding amount in the proliferative VSMCs was set at 1.0 in each experiment. Values are the mean ± standard error for three independent experiments.
    Figure Legend Snippet: The amount of GATA-6-DNA binding is up-regulated by induction of the differentiated phenotype. (A) Nuclear extracts were obtained from VSMCs with proliferative (P) or differentiated (D) phenotype. These extracts were probed with a radiolabeled oligonucleotide containing the Sm-MHC GATA site. Unlabeled competitor DNAs were present at a 100-fold molar excess as indicated: (lane 4) wild-type Sm-MHC GATA (wt); (lane 5) Sm-MHC GATA with a mutation (mut). The arrow indicates the complex corresponding to the GATA-specific interaction between the Sm-MHC GATA site and GATA-6. B, EMSA studies using the Sm-MHC-GATA probe were performed in nuclear extracts from VSMCs with differentiated (D) phenotype. Equal amounts of the anti–GATA-6 antibody (lane 3), the anti–GATA-4 antibody (lane 2), or normal rabbit IgG (lane 1) were added to the binding mixture. (C) EMSA studies using the probe for Sp-1 were performed in nuclear extracts from VSMCs with proliferative (P) or differentiated (D) phenotype as indicated. The arrow indicates the complex corresponding to the interaction between the probe and Sp-1. (D) The amount of GATA-6-DNA binding in A (lanes 1–3) and that of SP-1-DNA binding in C was quantified by densitometry using NIH image 1.61 and the relative DNA binding amount (Sm-MHC GATA/SP-1) was determined. The relative DNA binding amount in the proliferative VSMCs was set at 1.0 in each experiment. Values are the mean ± standard error for three independent experiments.

    Techniques Used: Binding Assay, Mutagenesis

    23) Product Images from "Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2( SK2) in mouse cardiac myocytes, et al. Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes"

    Article Title: Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2( SK2) in mouse cardiac myocytes, et al. Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes

    Journal: Acta Physiologica (Oxford, England)

    doi: 10.1111/apha.12986

    Colocalizations of JP 2 and SK 2 channels in adult mouse cardiac myocytes and HEK 293 cells. Confocal images show double immunostaining with anti‐ JP 2 antibody (green) and anti‐ SK 2 antibody (red) in single isolated mouse atria (A) and ventricular (B) myocytes as well as HEK 293 cells (D) cotransfected with JP 2 and SK 2. Negative control experiments (Control) were performed with the secondary antibodies with anti‐rabbit‐IgG TRITC ‐conjugated and with anti‐mouse‐IgG FITC ‐conjugated in atria myocytes (C) and HEK 293 cells (E). Merge images show the colocalization of JP 2 and SK 2 channels near the Z ‐lines in the cardiac cells or on the surface membrane of HEK 293 cells. Scale bars, 10 μm
    Figure Legend Snippet: Colocalizations of JP 2 and SK 2 channels in adult mouse cardiac myocytes and HEK 293 cells. Confocal images show double immunostaining with anti‐ JP 2 antibody (green) and anti‐ SK 2 antibody (red) in single isolated mouse atria (A) and ventricular (B) myocytes as well as HEK 293 cells (D) cotransfected with JP 2 and SK 2. Negative control experiments (Control) were performed with the secondary antibodies with anti‐rabbit‐IgG TRITC ‐conjugated and with anti‐mouse‐IgG FITC ‐conjugated in atria myocytes (C) and HEK 293 cells (E). Merge images show the colocalization of JP 2 and SK 2 channels near the Z ‐lines in the cardiac cells or on the surface membrane of HEK 293 cells. Scale bars, 10 μm

    Techniques Used: Double Immunostaining, Isolation, Negative Control

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    Incubation:

    Article Title: Mutant Escherichia coli Heat-Labile Toxin B Subunit That Separates Toxoid-Mediated Signaling and Immunomodulatory Action from Trafficking and Delivery Functions
    Article Snippet: Subsequently, plates were washed with PBS-Tween and incubated with monoclonal antibody LDS16, specific for the EtxB pentamer (a kind gift from R. F. James, Department of Surgery, University of Leicester, Leicester, United Kingdom), diluted in PBS-Tween (1:1,500) and incubated for 1 h at 37°C. .. Plates were then washed with PBS-Tween and incubated with a horseradish peroxidase-conjugated goat antibody directed against mouse IgG (1:10,000) (Jackson ImmunoResearch Laboratories) for 1 h, 37°C, and then washed twice with PBS-Tween, and once with PBS. .. Bound B subunit was then detected by the addition of 50 mM phosphate buffer, pH 5.5, containing 0.02% o -phenylenediamine dihydrochloride (Sigma) and 0.006% perhydrol (Merck, Darmstadt, Germany).

    Article Title: N-WASP Is Essential for the Negative Regulation of B Cell Receptor Signaling
    Article Snippet: .. Flow cytometry B cells were incubated with biotinylated F(ab′)2 -goat anti-mouse IgG+M (10 µg/ml; Jackson ImmunoResearch) at 4°C and chased at 37°C . .. Biotin-F(ab′)2 –anti-IgG+M left on the cell surface after the chase was stained with PE-streptavidin and quantified using a flow cytometer.

    Article Title: Parp3 Negatively Regulates Immunoglobulin Class Switch Recombination
    Article Snippet: Ninety-six–well plates (Nunc) were coated with 5 μg/ml NP(23)-BSA to detect both low- and high-affinity antibodies or NP(4)-BSA (Biosearch Technologies Inc.) to detect high-affinity antibodies. .. Goat anti−mouse IgM and goat anti−mouse IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch) were incubated for 1 h at 37°C. .. Horseradish peroxydase activity was revealed with SigmaFast OPD substrate kit (Sigma-Aldrich).

    Labeling:

    Article Title: Evaluation of Targeting Efficiency of Joints with Anticollagen II Antibodies
    Article Snippet: .. In order to exclude the role of dye in the observed targeting efficiency, the dyes were swapped: Arthrogen was labeled with IRDye 680, and mouse IgG was labeled with IRDye 800. .. BALB/c mice were injected iv with 40 μg of antibody, and the accumulation in the main organs and joints was studied at 24 h and 22 days post-injection.

    Flow Cytometry:

    Article Title: N-WASP Is Essential for the Negative Regulation of B Cell Receptor Signaling
    Article Snippet: .. Flow cytometry B cells were incubated with biotinylated F(ab′)2 -goat anti-mouse IgG+M (10 µg/ml; Jackson ImmunoResearch) at 4°C and chased at 37°C . .. Biotin-F(ab′)2 –anti-IgG+M left on the cell surface after the chase was stained with PE-streptavidin and quantified using a flow cytometer.

    Cytometry:

    Article Title: N-WASP Is Essential for the Negative Regulation of B Cell Receptor Signaling
    Article Snippet: .. Flow cytometry B cells were incubated with biotinylated F(ab′)2 -goat anti-mouse IgG+M (10 µg/ml; Jackson ImmunoResearch) at 4°C and chased at 37°C . .. Biotin-F(ab′)2 –anti-IgG+M left on the cell surface after the chase was stained with PE-streptavidin and quantified using a flow cytometer.

    Affinity Purification:

    Article Title: Identification of twenty-three mutations in fission yeast Scap that constitutively activate SREBP *
    Article Snippet: .. We obtained yeast extract from BD Biosciences; Edinburgh minimal medium (EMM) and amino acids from Q-Biogene; oligonucleotides from Integrated DNA Technologies; HRP-conjugated, affinity-purified donkey anti-rabbit and anti-mouse IgG from Jackson ImmunoResearch; cholesterol (C6760) from Steraloids; hydroxypropyl-β-cyclodextrin (HPCD) from Cyclodextrin Technologies Development; peptide N -glycosidase F from New England Biolabs; mevalonate, compactin, trypsin (type I from bovine pancreas), soybean trypsin inhibitor, protease inhibitors (leupeptin, pepstatin A, aprotinin, and PMSF), lipoprotein-deficient serum (LPDS), and oleate from Sigma; fatty-acid free BSA from SeraCare Life Sciences; digitonin and N -acetyl-leucyl-leucyl-norleucinal (ALLN) from Calbiochem. .. We obtained anti-Myc 9E10 IgG from Santa Cruz, anti-Myc polyclonal IgG from Upstate, and anti-HSV monoclonal IgG from Novagen.

    Transfection:

    Article Title: The Neural Cell Adhesion Molecule L1 Potentiates Integrin-Dependent Cell Migration to Extracellular Matrix Proteins
    Article Snippet: B35 cells were transfected for transient expression with HA-tagged ERK2 plasmid together with L1 plasmids, and phosphorylation of immunoprecipitated HA-ERK2 protein was measured by Western blotting with anti-Active MAPK antibody against dually phosphorylated, activated ERKs as described ( ). .. At 36–40 hr after transfection, the medium was replaced with OptiMEM-I, and 8 hr later cells were treated with preformed complexes of either nonimmune mouse IgG or L1 antibody Neuro4 and F(ab′)2 fragments of secondary antibodies against Fc fragments of mouse IgG (Jackson ImmunoResearch, West Grove, PA) for 10 min at 37°C. ..

    other:

    Article Title: Pseudotyping of HIV-1 with Human T-Lymphotropic Virus 1 (HTLV-1) Envelope Glycoprotein during HIV-1–HTLV-1 Coinfection Facilitates Direct HIV-1 Infection of Female Genital Epithelial Cells: Implications for Sexual Transmission of HIV-1
    Article Snippet: Mitomycin C-treated HIV-1–HTLV-1-coinfected T cells were preincubated with appropriate concentrations of antibodies as described previously ( , ) (PPH1, PHR4, PHR7A, and mouse IgG at 10 μg/ml; HAM anti-HTLV-1 IgG and normal human IgG at 100 μg/ml; 2G12 at 16 μg/ml).

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    Jackson Immuno peroxidase affinipure goat anti mouse igg fcγ subclass 1 specific
    Antibody and T-cell responses after GX-19 vaccination in macaques. Macaques (n=3) were immunized with 3 mg of GX-19 as described in the methods. Serum and PBMCs were collected before (wk 0), during (wk 4, and 5.5) and after (wk 8) vaccination and were assessed for SARS-CoV-2 S-specific <t>IgG</t> antibodies by ELISA (A) and neutralizing antibodies against SARS-CoV-2 live-virus (B) . Data represent mean SEM of individual macaques, and dashed line indicate the assay limits of detection. The number of SARS-CoV-2 S-specific IFN-γ secreting cells in PBMCs was determined by IFN-γ ELISPOT assay after stimulation with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 PBMCS in triplicate wells (C) . The frequency of S-specific CD4 + or CD8 + T cells producing IFN-γ, TNF-α, or IL-2 was determined by intracellular cytokine staining assays stimulated with SARS-CoV-2 S peptide pools. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (D) .
    Peroxidase Affinipure Goat Anti Mouse Igg Fcγ Subclass 1 Specific, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno anti mouse igg cy3 secondary antibody
    Double fluorescence colocalization of SMI-32 and ChAT immunoreativity with other markers. A–C , Large SMI-32(+) neurons are of cortical origin. Twelve-day-old cortical cultures were labeled with CT before addition of BF cells for an additional 5 d. Two large SMI-32(+) neurons are demonstrated under fluorescence ( A ; using a <t>Cy3-linked</t> secondary antibody; red ), under visible light ( B ), and again under fluorescence to reveal the CT labeling ( C ; using a fluorescein optical filter; green ). Note that the large SMI-32(+) neurons ( arrows ) are also labeled with the CT. D–I , Large SMI-32(+) neurons are not labeled by 192 <t>IgG–Cy3.</t> Photomicrographs show fields of BF–Cor cocultures that were grown for 18–20 d and then treated with 192 IgG–Cy3 hours before fixation and staining for ChAT ( D–F ) or SMI-32 ( G–I ). ChAT(+) and SMI-32(+) ( Figure legend continues ). neurons are viewed under fluorescence ( D , G , H ) using a Cy2-linked secondary antibody and a fluorescein optical filter ( green ), with a double exposure showing both Cy2 and Cy3 fluorescence ( E ), and with only a Cy3 optical filter to show 192 IgG–Cy3 accumulation ( F , I ; red ). Note that the ChAT(+) but not the SMI-32(+) neuron is clearly labeled with the 192 IgG–Cy3. The scattered areas of fluorescence in F and I are nonspecific clumps of stain and do not represent neurons. Scale bars: A–C , G , 100 μm; D–F , H , I , 50 μm.
    Anti Mouse Igg Cy3 Secondary Antibody, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno goat anti mouse igg m
    N-WASP is activated following WASP activation upon antigen stimulation. (A–D) TIRFM and IRM analysis of pWASP and pN-WASP in the B-cell contact zone of mouse splenic B cells and human PBMC B cells that were incubated with membrane-tethered AF546–mB-Fab′–anti-mouse or human <t>IgG+M</t> at 37°C for indicated times. (E and G) The MFI of pWASP or pN-WASP in the B-cell contact zone was quantified using TIRFM images and Andor iQ software. (F and H) The MFI of pWASP or pN-WASP in mouse splenic and human PBMC B cells incubated with soluble Fab′–anti-IgG+M plus streptavidin at 37°C for indicated times were analyzed by flow <t>cytometry.</t> Shown are representative images and the average MFI (±SD) from three independent experiments. Bar, 2.5 µm.
    Goat Anti Mouse Igg M, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno goat antimouse igg h l antibody
    Physicochemical properties of the MERS‐CoV nanoparticle vaccine. A) Size and zeta potential of adjuvant‐loaded nanoparticles before and after the MERS‐CoV RBD antigen conjugation. B) Estimated numbers of MERS‐CoV RBD antigens on each PLGA hollow nanoparticle. Nanoparticle‐attached antigens were calculated by directly quantifying protein contents on nanoparticles after conjugation reaction using the BCA protein assay. C) Loading of cdGMP in synthetic hollow nanoparticles before and after conjugation with recombinant MERS‐CoV RBD antigens. D) Cryo‐electron microscopy and E) transmission electron microscopy of MERS‐CoV RBD coated nanoparticles. F) Immunogold staining of the MERS‐CoV RBD conjugated nanoparticle with anti‐His tag and goat <t>antimouse</t> <t>IgG</t> antibodies followed by transmission electron microscopy. Error bars represent mean ± SEM ( N = 3).
    Goat Antimouse Igg H L Antibody, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Antibody and T-cell responses after GX-19 vaccination in macaques. Macaques (n=3) were immunized with 3 mg of GX-19 as described in the methods. Serum and PBMCs were collected before (wk 0), during (wk 4, and 5.5) and after (wk 8) vaccination and were assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (A) and neutralizing antibodies against SARS-CoV-2 live-virus (B) . Data represent mean SEM of individual macaques, and dashed line indicate the assay limits of detection. The number of SARS-CoV-2 S-specific IFN-γ secreting cells in PBMCs was determined by IFN-γ ELISPOT assay after stimulation with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 PBMCS in triplicate wells (C) . The frequency of S-specific CD4 + or CD8 + T cells producing IFN-γ, TNF-α, or IL-2 was determined by intracellular cytokine staining assays stimulated with SARS-CoV-2 S peptide pools. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (D) .

    Journal: bioRxiv

    Article Title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

    doi: 10.1101/2020.10.09.334136

    Figure Lengend Snippet: Antibody and T-cell responses after GX-19 vaccination in macaques. Macaques (n=3) were immunized with 3 mg of GX-19 as described in the methods. Serum and PBMCs were collected before (wk 0), during (wk 4, and 5.5) and after (wk 8) vaccination and were assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (A) and neutralizing antibodies against SARS-CoV-2 live-virus (B) . Data represent mean SEM of individual macaques, and dashed line indicate the assay limits of detection. The number of SARS-CoV-2 S-specific IFN-γ secreting cells in PBMCs was determined by IFN-γ ELISPOT assay after stimulation with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 PBMCS in triplicate wells (C) . The frequency of S-specific CD4 + or CD8 + T cells producing IFN-γ, TNF-α, or IL-2 was determined by intracellular cytokine staining assays stimulated with SARS-CoV-2 S peptide pools. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (D) .

    Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205), or IgG2a (Jackson ImmunoResearch Laboratories 115-035-206) or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P) for the NHP sera for 1 hour at 37°C.

    Techniques: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Staining

    Immunization with GX-19 elicit Th1-biased T cell responses in mice. BALB/c mice (n=3-7/group) were immunized at week 0 and 2 with indicated doses of GX-19 or pGX27 (empty control vector) as described in the methods (A-C) . Sera were collected 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG1 and IgG2a/b. Endpoint titers (A) , and endpoint tier ratios of IgG2a/b to IgG1 (B) were calculated. 2 weeks post-boost mouse splenocytes were isolated and re-stimulated with peptide pools spanning the SARS-CoV-2 S protein ex vivo . Indicated cytokines in the supernatants of culture were quantified using Th1/Th2 cytometric bead array kit (D) . T cell responses were measured by IFN-γ ELISPOT in splenocytes stimulated with peptide pools spanning the SARS-CoV-2 S protein (C) . Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (E) . Data representative of two independent experiments. P values determined by Mann-Whitney test.

    Journal: bioRxiv

    Article Title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

    doi: 10.1101/2020.10.09.334136

    Figure Lengend Snippet: Immunization with GX-19 elicit Th1-biased T cell responses in mice. BALB/c mice (n=3-7/group) were immunized at week 0 and 2 with indicated doses of GX-19 or pGX27 (empty control vector) as described in the methods (A-C) . Sera were collected 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG1 and IgG2a/b. Endpoint titers (A) , and endpoint tier ratios of IgG2a/b to IgG1 (B) were calculated. 2 weeks post-boost mouse splenocytes were isolated and re-stimulated with peptide pools spanning the SARS-CoV-2 S protein ex vivo . Indicated cytokines in the supernatants of culture were quantified using Th1/Th2 cytometric bead array kit (D) . T cell responses were measured by IFN-γ ELISPOT in splenocytes stimulated with peptide pools spanning the SARS-CoV-2 S protein (C) . Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (E) . Data representative of two independent experiments. P values determined by Mann-Whitney test.

    Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205), or IgG2a (Jackson ImmunoResearch Laboratories 115-035-206) or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P) for the NHP sera for 1 hour at 37°C.

    Techniques: Mouse Assay, Plasmid Preparation, Isolation, Ex Vivo, Enzyme-linked Immunospot, Staining, MANN-WHITNEY

    Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) (A) . BALB/c mice (n=4-10/group) were immunized at week 0 and 2 with pGX27-S ΔTM , pGX27-S or pGX27 (empty control vector) as described in the methods. Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies (B) .

    Journal: bioRxiv

    Article Title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

    doi: 10.1101/2020.10.09.334136

    Figure Lengend Snippet: Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) (A) . BALB/c mice (n=4-10/group) were immunized at week 0 and 2 with pGX27-S ΔTM , pGX27-S or pGX27 (empty control vector) as described in the methods. Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies (B) .

    Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205), or IgG2a (Jackson ImmunoResearch Laboratories 115-035-206) or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P) for the NHP sera for 1 hour at 37°C.

    Techniques: Expressing, Mouse Assay, Plasmid Preparation

    GX-19 elicit robust binding and neutralizing antibody responses in mice. BALB/c mice (n=4-7/group) were immunized at week 0 and 2 with indicated doses of GX-19 or pGX27 as described in the methods (A-C) . Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (A) , and for post-boost sera, neutralizing antibodies against SARS-CoV-2 live-virus (C) . BAL were collected 2 weeks post-boost and assayed for SARS-CoV-2 S-specific IgG antibodies by ELISA (B) . Data representative of two independent experiments. P values determined by Mann-Whitney test.

    Journal: bioRxiv

    Article Title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

    doi: 10.1101/2020.10.09.334136

    Figure Lengend Snippet: GX-19 elicit robust binding and neutralizing antibody responses in mice. BALB/c mice (n=4-7/group) were immunized at week 0 and 2 with indicated doses of GX-19 or pGX27 as described in the methods (A-C) . Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (A) , and for post-boost sera, neutralizing antibodies against SARS-CoV-2 live-virus (C) . BAL were collected 2 weeks post-boost and assayed for SARS-CoV-2 S-specific IgG antibodies by ELISA (B) . Data representative of two independent experiments. P values determined by Mann-Whitney test.

    Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205), or IgG2a (Jackson ImmunoResearch Laboratories 115-035-206) or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P) for the NHP sera for 1 hour at 37°C.

    Techniques: Binding Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Double fluorescence colocalization of SMI-32 and ChAT immunoreativity with other markers. A–C , Large SMI-32(+) neurons are of cortical origin. Twelve-day-old cortical cultures were labeled with CT before addition of BF cells for an additional 5 d. Two large SMI-32(+) neurons are demonstrated under fluorescence ( A ; using a Cy3-linked secondary antibody; red ), under visible light ( B ), and again under fluorescence to reveal the CT labeling ( C ; using a fluorescein optical filter; green ). Note that the large SMI-32(+) neurons ( arrows ) are also labeled with the CT. D–I , Large SMI-32(+) neurons are not labeled by 192 IgG–Cy3. Photomicrographs show fields of BF–Cor cocultures that were grown for 18–20 d and then treated with 192 IgG–Cy3 hours before fixation and staining for ChAT ( D–F ) or SMI-32 ( G–I ). ChAT(+) and SMI-32(+) ( Figure legend continues ). neurons are viewed under fluorescence ( D , G , H ) using a Cy2-linked secondary antibody and a fluorescein optical filter ( green ), with a double exposure showing both Cy2 and Cy3 fluorescence ( E ), and with only a Cy3 optical filter to show 192 IgG–Cy3 accumulation ( F , I ; red ). Note that the ChAT(+) but not the SMI-32(+) neuron is clearly labeled with the 192 IgG–Cy3. The scattered areas of fluorescence in F and I are nonspecific clumps of stain and do not represent neurons. Scale bars: A–C , G , 100 μm; D–F , H , I , 50 μm.

    Journal: The Journal of Neuroscience

    Article Title: Distinctive Morphological Features of a Subset of Cortical Neurons Grown in the Presence of Basal Forebrain Neurons In Vitro

    doi: 10.1523/JNEUROSCI.18-11-04201.1998

    Figure Lengend Snippet: Double fluorescence colocalization of SMI-32 and ChAT immunoreativity with other markers. A–C , Large SMI-32(+) neurons are of cortical origin. Twelve-day-old cortical cultures were labeled with CT before addition of BF cells for an additional 5 d. Two large SMI-32(+) neurons are demonstrated under fluorescence ( A ; using a Cy3-linked secondary antibody; red ), under visible light ( B ), and again under fluorescence to reveal the CT labeling ( C ; using a fluorescein optical filter; green ). Note that the large SMI-32(+) neurons ( arrows ) are also labeled with the CT. D–I , Large SMI-32(+) neurons are not labeled by 192 IgG–Cy3. Photomicrographs show fields of BF–Cor cocultures that were grown for 18–20 d and then treated with 192 IgG–Cy3 hours before fixation and staining for ChAT ( D–F ) or SMI-32 ( G–I ). ChAT(+) and SMI-32(+) ( Figure legend continues ). neurons are viewed under fluorescence ( D , G , H ) using a Cy2-linked secondary antibody and a fluorescein optical filter ( green ), with a double exposure showing both Cy2 and Cy3 fluorescence ( E ), and with only a Cy3 optical filter to show 192 IgG–Cy3 accumulation ( F , I ; red ). Note that the ChAT(+) but not the SMI-32(+) neuron is clearly labeled with the 192 IgG–Cy3. The scattered areas of fluorescence in F and I are nonspecific clumps of stain and do not represent neurons. Scale bars: A–C , G , 100 μm; D–F , H , I , 50 μm.

    Article Snippet: Again, an anti-mouse IgG–Cy3 secondary antibody was used to visualize cells labeled with the SMI-32 primary antibody.

    Techniques: Fluorescence, Labeling, Staining

    N-WASP is activated following WASP activation upon antigen stimulation. (A–D) TIRFM and IRM analysis of pWASP and pN-WASP in the B-cell contact zone of mouse splenic B cells and human PBMC B cells that were incubated with membrane-tethered AF546–mB-Fab′–anti-mouse or human IgG+M at 37°C for indicated times. (E and G) The MFI of pWASP or pN-WASP in the B-cell contact zone was quantified using TIRFM images and Andor iQ software. (F and H) The MFI of pWASP or pN-WASP in mouse splenic and human PBMC B cells incubated with soluble Fab′–anti-IgG+M plus streptavidin at 37°C for indicated times were analyzed by flow cytometry. Shown are representative images and the average MFI (±SD) from three independent experiments. Bar, 2.5 µm.

    Journal: PLoS Biology

    Article Title: N-WASP Is Essential for the Negative Regulation of B Cell Receptor Signaling

    doi: 10.1371/journal.pbio.1001704

    Figure Lengend Snippet: N-WASP is activated following WASP activation upon antigen stimulation. (A–D) TIRFM and IRM analysis of pWASP and pN-WASP in the B-cell contact zone of mouse splenic B cells and human PBMC B cells that were incubated with membrane-tethered AF546–mB-Fab′–anti-mouse or human IgG+M at 37°C for indicated times. (E and G) The MFI of pWASP or pN-WASP in the B-cell contact zone was quantified using TIRFM images and Andor iQ software. (F and H) The MFI of pWASP or pN-WASP in mouse splenic and human PBMC B cells incubated with soluble Fab′–anti-IgG+M plus streptavidin at 37°C for indicated times were analyzed by flow cytometry. Shown are representative images and the average MFI (±SD) from three independent experiments. Bar, 2.5 µm.

    Article Snippet: Flow cytometry B cells were incubated with biotinylated F(ab′)2 -goat anti-mouse IgG+M (10 µg/ml; Jackson ImmunoResearch) at 4°C and chased at 37°C .

    Techniques: Activation Assay, Incubation, Software, Flow Cytometry, Cytometry

    Physicochemical properties of the MERS‐CoV nanoparticle vaccine. A) Size and zeta potential of adjuvant‐loaded nanoparticles before and after the MERS‐CoV RBD antigen conjugation. B) Estimated numbers of MERS‐CoV RBD antigens on each PLGA hollow nanoparticle. Nanoparticle‐attached antigens were calculated by directly quantifying protein contents on nanoparticles after conjugation reaction using the BCA protein assay. C) Loading of cdGMP in synthetic hollow nanoparticles before and after conjugation with recombinant MERS‐CoV RBD antigens. D) Cryo‐electron microscopy and E) transmission electron microscopy of MERS‐CoV RBD coated nanoparticles. F) Immunogold staining of the MERS‐CoV RBD conjugated nanoparticle with anti‐His tag and goat antimouse IgG antibodies followed by transmission electron microscopy. Error bars represent mean ± SEM ( N = 3).

    Journal: Advanced Functional Materials

    Article Title: Viromimetic STING Agonist‐Loaded Hollow Polymeric Nanoparticles for Safe and Effective Vaccination against Middle East Respiratory Syndrome Coronavirus, Viromimetic STING Agonist‐Loaded Hollow Polymeric Nanoparticles for Safe and Effective Vaccination against Middle East Respiratory Syndrome Coronavirus

    doi: 10.1002/adfm.201807616

    Figure Lengend Snippet: Physicochemical properties of the MERS‐CoV nanoparticle vaccine. A) Size and zeta potential of adjuvant‐loaded nanoparticles before and after the MERS‐CoV RBD antigen conjugation. B) Estimated numbers of MERS‐CoV RBD antigens on each PLGA hollow nanoparticle. Nanoparticle‐attached antigens were calculated by directly quantifying protein contents on nanoparticles after conjugation reaction using the BCA protein assay. C) Loading of cdGMP in synthetic hollow nanoparticles before and after conjugation with recombinant MERS‐CoV RBD antigens. D) Cryo‐electron microscopy and E) transmission electron microscopy of MERS‐CoV RBD coated nanoparticles. F) Immunogold staining of the MERS‐CoV RBD conjugated nanoparticle with anti‐His tag and goat antimouse IgG antibodies followed by transmission electron microscopy. Error bars represent mean ± SEM ( N = 3).

    Article Snippet: For nanoparticle visualization, negative staining was performed with uranyl acetate and immunogold staining was performed using mouse anti‐His tag antibody (Roche) and goat antimouse IgG (H+L) antibody conjugated with 6 nm colloidal gold (Jackson ImmunoResearch, West Grove, PA).

    Techniques: Conjugation Assay, Bicinchoninic Acid Protein Assay, Recombinant, Electron Microscopy, Transmission Assay, Staining