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    Name:
    Dynabeads Protein G for Immunoprecipitation
    Description:
    Dynabeads Protein G are uniform 2 8 µm superparamagnetic beads with recombinant Protein G 17 kDa covalently coupled to the surface Dynabeads Protein G provide a superior alternative to Sepharose or agarose slurry for immunoprecipitation IP and both manual and automated protocols are available • IP in less than 40 minutes • High target protein yield with low antibody consumption • Very low non specific binding with high signal to noise ratio • No columns centrifugations or time consuming pre clearing required • High reproducibility and high throughput compatible with KingFisher instruments Manual Dynabeads separation is fast and easy to perform The manual protocol is simple and can be performed in under 40 minutes First the antibody for the target protein is incubated with the Dynabeads Protein G in a tube for 10 minutes Excess antibody is washed away by placing the tube in a DynaMag magnet and removing the supernatant The antibody coated beads can then be used for a variety of downstream applications including IP Co IP chromatin IP ChIP RNA IP RIP small scale IgG purification and protein purification Bound material is easily collected using a DynaMag magnet due to the unique magnetic properties of the Dynabeads The recombinant protein G on the beads contains no albumin binding sites thus albumin is not co purified during the procedure The IP is fast and gives high yield high reproducibility and very little non specific binding thus pre clearing is not required Automated Dynabeads separation helps increase throughput and reduces hands on time If you are working with several samples in parallel the number of washing steps and the hands on time increases proportionally with the number of samples Pipetting and other manual handling tend to be less consistent than automation when working with many samples at a time To better handle a medium to high throughput number of samples reduce hands on time and secure high reproducibility we have developed IP protocols for the KingFisher Flex and KingFisher Duo Prime instruments The automated protocols replicate the manual protocols obtaining equally high target protein yield and the same low non specific binding and high reproducibility It doesn t matter if you are working with 10 or 96 samples the IP protocol is less than 40 minutes regardless Just load the reagents on the plates push the “Start button and by the time you have prepared for downstream analysis the IP is done Some optimization e g incubation times might be necessary depending on your antibody and the abundance and or specificity of your target protein • Use the KingFisher Duo instrument for low to medium throughput 1 12 samples run • Use the KingFisher Flex instrument for high throughput 12 96 samples run See automated protocols Watch a video about the KingFisher Flex instrument Gentle separation causes minimal physical stress to proteins The magnetic separation technology utilized by Dynabeads Protein G is rapid and gentle causing minimal physical stress to your target proteins This permits the isolation and concentration of labile composites that might otherwise dissociate or be damaged by proteases during long incubation times Native protein conformation and large protein complexes are preserved Binding strength and capacity Dynabeads Protein G allow for isolation of most mammalian immunoglobulins Ig The amount of Ig captured depends on the concentration of Ig in the starting sample and on the type and source of the Ig 100 µL of Dynabeads Protein G will isolate approximately 25 30 µg human IgG from a sample containing 20 200 µg IgG mL Predominant Fc binding allows optimal Ig orientation The antibodies bind to the outer smooth surface of the beads thus are not trapped in large pores as with Sepharose agarose based beads All antibodies are available for protein binding so low amounts of antibody are required while still obtaining the same high yield of target protein The smooth bead surface is also responsible for the low non specific binding that Dynabeads are known for Learn more about Dynabeads • Dynabeads Protein A are also available as a ready to go kit with buffers included • See immunoprecipitation selection guides data and references • See magnets for Dynabeads separations • Find Dynabeads products for other applications OEM purchase To purchase Dynabeads Protein A and Protein G on an OEM basis contact our Out Licensing and OEM Sales department Sepharose is a trademark of GE Healthcare Bio Sciences AB
    Catalog Number:
    10003d
    Price:
    None
    Applications:
    Antibody Production and Labeling|Antibody Purification|Cell Lysis & Fractionation|ChIP-on-Chip|Chromatin Immunoprecipitation (ChIP)|Immunoprecipitation|Organelle Isolation|Phage Display|Protein Assays and Analysis|Protein Biology|Protein Complex Isolation & Analysis|Protein Microarrays|Protein Purification|Protein Purification & Isolation|Protein-Protein Interactions|RNAi, Epigenetics & Non-Coding RNA Research|Chromatin Biology
    Category:
    Beads Microspheres
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    Structured Review

    Thermo Fisher scrsh et
    Regulation of the MYB promoter by ZEB1. (A) (i) Outline of several CAT reporter constructs containing MYB promoter and intron 1 sequences with various numbers of E and Z boxes, which were used in the CAT ELISA assay. (ii) Western blot of HEK 293 cells transfected with various amounts of pcDNA3.1-ZEB1 expression construct, as surrogate validation of ZEB1 expression in the CAT ELISA assay. (iii) Results of a CAT ELISA assay, “+ZEB1” expressed as fold change from each individual CAT reporter alone, n = 1; error bars represent standard deviation. (B) Scheme of the MYB gene indicating (black bars) the position of E-boxes and Z-boxes, sites at which ZEB1 may bind [ 4 ]. Red bars indicate QRT-PCR primers used in the ChIP assay. (C) ChIP analysis of <t>SCRsh-ET</t> and <t>ZEB1sh-ET</t> cells using anti-ZEB1 (E20; Santa Cruz) and control anti-goat IgG. The graph depicts the enrichment of PCR-amplified immunoprecipitated DNA expressed as a percentage of total DNA immunoprecipitated with ZEB1 antibody or control anti-goat IgG relative to unprecipitated input sample. Data are representative of three independent assays (error bars represent SD). Additional data are shown in Additional file 9 : Figure S9B.
    Dynabeads Protein G are uniform 2 8 µm superparamagnetic beads with recombinant Protein G 17 kDa covalently coupled to the surface Dynabeads Protein G provide a superior alternative to Sepharose or agarose slurry for immunoprecipitation IP and both manual and automated protocols are available • IP in less than 40 minutes • High target protein yield with low antibody consumption • Very low non specific binding with high signal to noise ratio • No columns centrifugations or time consuming pre clearing required • High reproducibility and high throughput compatible with KingFisher instruments Manual Dynabeads separation is fast and easy to perform The manual protocol is simple and can be performed in under 40 minutes First the antibody for the target protein is incubated with the Dynabeads Protein G in a tube for 10 minutes Excess antibody is washed away by placing the tube in a DynaMag magnet and removing the supernatant The antibody coated beads can then be used for a variety of downstream applications including IP Co IP chromatin IP ChIP RNA IP RIP small scale IgG purification and protein purification Bound material is easily collected using a DynaMag magnet due to the unique magnetic properties of the Dynabeads The recombinant protein G on the beads contains no albumin binding sites thus albumin is not co purified during the procedure The IP is fast and gives high yield high reproducibility and very little non specific binding thus pre clearing is not required Automated Dynabeads separation helps increase throughput and reduces hands on time If you are working with several samples in parallel the number of washing steps and the hands on time increases proportionally with the number of samples Pipetting and other manual handling tend to be less consistent than automation when working with many samples at a time To better handle a medium to high throughput number of samples reduce hands on time and secure high reproducibility we have developed IP protocols for the KingFisher Flex and KingFisher Duo Prime instruments The automated protocols replicate the manual protocols obtaining equally high target protein yield and the same low non specific binding and high reproducibility It doesn t matter if you are working with 10 or 96 samples the IP protocol is less than 40 minutes regardless Just load the reagents on the plates push the “Start button and by the time you have prepared for downstream analysis the IP is done Some optimization e g incubation times might be necessary depending on your antibody and the abundance and or specificity of your target protein • Use the KingFisher Duo instrument for low to medium throughput 1 12 samples run • Use the KingFisher Flex instrument for high throughput 12 96 samples run See automated protocols Watch a video about the KingFisher Flex instrument Gentle separation causes minimal physical stress to proteins The magnetic separation technology utilized by Dynabeads Protein G is rapid and gentle causing minimal physical stress to your target proteins This permits the isolation and concentration of labile composites that might otherwise dissociate or be damaged by proteases during long incubation times Native protein conformation and large protein complexes are preserved Binding strength and capacity Dynabeads Protein G allow for isolation of most mammalian immunoglobulins Ig The amount of Ig captured depends on the concentration of Ig in the starting sample and on the type and source of the Ig 100 µL of Dynabeads Protein G will isolate approximately 25 30 µg human IgG from a sample containing 20 200 µg IgG mL Predominant Fc binding allows optimal Ig orientation The antibodies bind to the outer smooth surface of the beads thus are not trapped in large pores as with Sepharose agarose based beads All antibodies are available for protein binding so low amounts of antibody are required while still obtaining the same high yield of target protein The smooth bead surface is also responsible for the low non specific binding that Dynabeads are known for Learn more about Dynabeads • Dynabeads Protein A are also available as a ready to go kit with buffers included • See immunoprecipitation selection guides data and references • See magnets for Dynabeads separations • Find Dynabeads products for other applications OEM purchase To purchase Dynabeads Protein A and Protein G on an OEM basis contact our Out Licensing and OEM Sales department Sepharose is a trademark of GE Healthcare Bio Sciences AB
    https://www.bioz.com/result/scrsh et/product/Thermo Fisher
    Average 85 stars, based on 3922 article reviews
    Price from $9.99 to $1999.99
    scrsh et - by Bioz Stars, 2020-08
    85/100 stars

    Images

    1) Product Images from "Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells"

    Article Title: Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr3580

    Regulation of the MYB promoter by ZEB1. (A) (i) Outline of several CAT reporter constructs containing MYB promoter and intron 1 sequences with various numbers of E and Z boxes, which were used in the CAT ELISA assay. (ii) Western blot of HEK 293 cells transfected with various amounts of pcDNA3.1-ZEB1 expression construct, as surrogate validation of ZEB1 expression in the CAT ELISA assay. (iii) Results of a CAT ELISA assay, “+ZEB1” expressed as fold change from each individual CAT reporter alone, n = 1; error bars represent standard deviation. (B) Scheme of the MYB gene indicating (black bars) the position of E-boxes and Z-boxes, sites at which ZEB1 may bind [ 4 ]. Red bars indicate QRT-PCR primers used in the ChIP assay. (C) ChIP analysis of SCRsh-ET and ZEB1sh-ET cells using anti-ZEB1 (E20; Santa Cruz) and control anti-goat IgG. The graph depicts the enrichment of PCR-amplified immunoprecipitated DNA expressed as a percentage of total DNA immunoprecipitated with ZEB1 antibody or control anti-goat IgG relative to unprecipitated input sample. Data are representative of three independent assays (error bars represent SD). Additional data are shown in Additional file 9 : Figure S9B.
    Figure Legend Snippet: Regulation of the MYB promoter by ZEB1. (A) (i) Outline of several CAT reporter constructs containing MYB promoter and intron 1 sequences with various numbers of E and Z boxes, which were used in the CAT ELISA assay. (ii) Western blot of HEK 293 cells transfected with various amounts of pcDNA3.1-ZEB1 expression construct, as surrogate validation of ZEB1 expression in the CAT ELISA assay. (iii) Results of a CAT ELISA assay, “+ZEB1” expressed as fold change from each individual CAT reporter alone, n = 1; error bars represent standard deviation. (B) Scheme of the MYB gene indicating (black bars) the position of E-boxes and Z-boxes, sites at which ZEB1 may bind [ 4 ]. Red bars indicate QRT-PCR primers used in the ChIP assay. (C) ChIP analysis of SCRsh-ET and ZEB1sh-ET cells using anti-ZEB1 (E20; Santa Cruz) and control anti-goat IgG. The graph depicts the enrichment of PCR-amplified immunoprecipitated DNA expressed as a percentage of total DNA immunoprecipitated with ZEB1 antibody or control anti-goat IgG relative to unprecipitated input sample. Data are representative of three independent assays (error bars represent SD). Additional data are shown in Additional file 9 : Figure S9B.

    Techniques Used: Construct, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Expressing, Standard Deviation, Quantitative RT-PCR, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Immunoprecipitation

    ZEB1sh-ET cells are more epithelial, express higher MYB, and are more proliferative than SCRsh-ET controls. (A) (i) Western blotting for ZEB1 and CDH1 in PMC42-ET cells transfected with shRNA variants 1 to 4; (ii) bar graph of band intensity of the Western blot shown in (i) . (iii) Expression (MT-PCR) of EMT-related genes, ZEB1sh-ET relative to SCRsh-ET; fold expression shown. Data shown are the average of four independent biologic replicates; Student paired t test was used to determine significance (*), set at P
    Figure Legend Snippet: ZEB1sh-ET cells are more epithelial, express higher MYB, and are more proliferative than SCRsh-ET controls. (A) (i) Western blotting for ZEB1 and CDH1 in PMC42-ET cells transfected with shRNA variants 1 to 4; (ii) bar graph of band intensity of the Western blot shown in (i) . (iii) Expression (MT-PCR) of EMT-related genes, ZEB1sh-ET relative to SCRsh-ET; fold expression shown. Data shown are the average of four independent biologic replicates; Student paired t test was used to determine significance (*), set at P

    Techniques Used: Western Blot, Transfection, shRNA, Expressing, Polymerase Chain Reaction

    2) Product Images from "Self-antigen recognition by follicular lymphoma B-cell receptors"

    Article Title: Self-antigen recognition by follicular lymphoma B-cell receptors

    Journal: Blood

    doi: 10.1182/blood-2012-05-427534

    Identification of myoferlin as a uniquely recognized self-antigen. (A) Silver stain of a 3%-8% Tris-acetate gel of proteins immunoprecipitated (IP) from HEp-2 cell lysate by the indicated recombinant tumor Igs. (−) indicates lanes containing tumor Ig proteins only; IP, lanes containing the immunoprecipitated proteins; Ly, lysate; and B, lysate IP with protein G beads only. The left arrow indicates the 236-kDa protein immunoprecipitated by the tumor Ig of patient 1152; the right arrow points to the location of the tumor Igs. (B) Immunoblotting for myoferlin in immunoprecipitation samples from HEp-2 cell lysate. (C) Immunoblotting for myoferlin in immunoprecipitation samples from 293T cells transfected with recombinant myoferlin. (D) A total of 98 tumor Igs were tested for binding to recombinant myoferlin by ELISA. Myoferlin-HA was immobilized using anti-HA antibodies on lysates from untransfected (left panel) and transfected 293T cells (right panel). Shown is a representative graph of OD 405-490 values for 14 different nonbinding patients' tumor Igs (solid lines) compared with tumor Ig for patient 1152 (dotted line). The ability of tumor Ig 1152 to bind myoferlin was confirmed in at least 2 independent experiments.
    Figure Legend Snippet: Identification of myoferlin as a uniquely recognized self-antigen. (A) Silver stain of a 3%-8% Tris-acetate gel of proteins immunoprecipitated (IP) from HEp-2 cell lysate by the indicated recombinant tumor Igs. (−) indicates lanes containing tumor Ig proteins only; IP, lanes containing the immunoprecipitated proteins; Ly, lysate; and B, lysate IP with protein G beads only. The left arrow indicates the 236-kDa protein immunoprecipitated by the tumor Ig of patient 1152; the right arrow points to the location of the tumor Igs. (B) Immunoblotting for myoferlin in immunoprecipitation samples from HEp-2 cell lysate. (C) Immunoblotting for myoferlin in immunoprecipitation samples from 293T cells transfected with recombinant myoferlin. (D) A total of 98 tumor Igs were tested for binding to recombinant myoferlin by ELISA. Myoferlin-HA was immobilized using anti-HA antibodies on lysates from untransfected (left panel) and transfected 293T cells (right panel). Shown is a representative graph of OD 405-490 values for 14 different nonbinding patients' tumor Igs (solid lines) compared with tumor Ig for patient 1152 (dotted line). The ability of tumor Ig 1152 to bind myoferlin was confirmed in at least 2 independent experiments.

    Techniques Used: Silver Staining, Immunoprecipitation, Recombinant, Transfection, Binding Assay, Enzyme-linked Immunosorbent Assay

    Igs of 1152 tumor subclones retain self-reactivity and antigen binding. (A) HEp-2 IFA staining pattern of tumor subclone Igs (2E12, 6C12, 4B11, 1G2, and 1E9) were obtained through rescue fusion of cells from the tumor biopsy of patient 1152; 1152 corresponds to the recombinant tumor Ig from patient 1152. Bars represent 25 μm. (B) Immunoblot for myoferlin in immunoprecipitations from lysate of 293T cells transfected with recombinant myoferlin-HA construct. Ly indicates lysate; B, lysate IP with protein G beads only; and 0516, an unrelated tumor Ig. (C) Tumor subclone Igs were tested for binding to recombinant myoferlin by ELISA. Recombinant Myoferlin-HA protein was immobilized using anti-HA antibodies on lysate from transfected 293T cells. Data shown are representative of at least 2 independent experiments.
    Figure Legend Snippet: Igs of 1152 tumor subclones retain self-reactivity and antigen binding. (A) HEp-2 IFA staining pattern of tumor subclone Igs (2E12, 6C12, 4B11, 1G2, and 1E9) were obtained through rescue fusion of cells from the tumor biopsy of patient 1152; 1152 corresponds to the recombinant tumor Ig from patient 1152. Bars represent 25 μm. (B) Immunoblot for myoferlin in immunoprecipitations from lysate of 293T cells transfected with recombinant myoferlin-HA construct. Ly indicates lysate; B, lysate IP with protein G beads only; and 0516, an unrelated tumor Ig. (C) Tumor subclone Igs were tested for binding to recombinant myoferlin by ELISA. Recombinant Myoferlin-HA protein was immobilized using anti-HA antibodies on lysate from transfected 293T cells. Data shown are representative of at least 2 independent experiments.

    Techniques Used: Binding Assay, Immunofluorescence, Staining, Recombinant, Transfection, Construct, Enzyme-linked Immunosorbent Assay

    3) Product Images from "Specific antibody-receptor interactions trigger InlAB-independent uptake of listeria monocytogenes into tumor cell lines"

    Article Title: Specific antibody-receptor interactions trigger InlAB-independent uptake of listeria monocytogenes into tumor cell lines

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-11-163

    Internalization of antibody coated Dynabeads Protein A into 4T1-HER2 cells . The beads were coated with the first antibody (1) and incubated with 4T1-HER2 cells. Following washing, the cells were incubated with the second antibody (2) and analyzed by confocal immunofluorescence microscopy. Beads labeled with (1) are located intracellular, while beads labeled with (1) and (2) are located extracellular. Non coated beads showed no background fluorescence (I) and were efficiently coated with Trastuzumab-Alexa488. On bead-coating with Trastuzumab or Trastuzumab-Alexa488 (II, III) some beads were located in the cell (marked with white arrowheads). Some beads remained outside the cells (marked with black arrowheads). Presence of bead fluorescence was analyzed in image stacks of at least 5 μm thickness to exclude false negatives (Additional file 4 ).
    Figure Legend Snippet: Internalization of antibody coated Dynabeads Protein A into 4T1-HER2 cells . The beads were coated with the first antibody (1) and incubated with 4T1-HER2 cells. Following washing, the cells were incubated with the second antibody (2) and analyzed by confocal immunofluorescence microscopy. Beads labeled with (1) are located intracellular, while beads labeled with (1) and (2) are located extracellular. Non coated beads showed no background fluorescence (I) and were efficiently coated with Trastuzumab-Alexa488. On bead-coating with Trastuzumab or Trastuzumab-Alexa488 (II, III) some beads were located in the cell (marked with white arrowheads). Some beads remained outside the cells (marked with black arrowheads). Presence of bead fluorescence was analyzed in image stacks of at least 5 μm thickness to exclude false negatives (Additional file 4 ).

    Techniques Used: Incubation, Immunofluorescence, Microscopy, Labeling, Fluorescence

    4) Product Images from "A glycogenin homolog controls Toxoplasma gondii growth via glycosylation of an E3 ubiquitin ligase"

    Article Title: A glycogenin homolog controls Toxoplasma gondii growth via glycosylation of an E3 ubiquitin ligase

    Journal: bioRxiv

    doi: 10.1101/764241

    Substrate specificity of Gat1. ( A ) Recombinantly expressed and purified preparations of TgGat1 and PuGat1 were analyzed by SDS-PAGE and staining with Coomassie blue. ( B ) Temporal dependence of UDP-Gal and UDP-Glc hydrolysis. The averages and standard deviations of 3 technical replicates are shown. A similar profile was observed with a different enzyme concentration. See Fig. S9E for a trial with higher enzyme concentrations. ( C ) Transferase activity utilizing 8 µM UDP-Gal or UDP-Glc toward 20 mM maltose-pNP for TgGat1 and PuGat1. The averages and standard deviations of two technical replicates are shown; similar profiles were in 2 independent assays with a different TgGat1 preparation. ( D ) UDP-Gal and UDP-Glc concentration dependence of TgGat1 transferase activity toward 20 mM maltose-pNP. The averages and standard deviations of two technical replicates are shown, and an independent trial with TgGat1 and PuGat1 against UDP-Gal is shown in Fig S9F. ( E ) Maltose-pNP concentration dependence of TgGat1 and PuGat1 transferase activity from 20 µM UDP-Gal. The averages and standard deviations of two technical replicates are shown. ( F ) Relative Gal-transferase activity of TgGat1 and PuGat1 toward different acceptors. The averages and standard deviations of three technical replicates are shown. Similar results were obtained in three independent trials. ( G ) Effect of UDP-Glc concentration on the Gal-transferase activity of TgGat1. Reactions were incubated for 1 h. The averages and standard deviations of two technical replicates are shown. (H ) Gal-transferase activity of TgGaT1 toward varied concentrations of GlFGaGn-Skp1, in the presence of 40 µM UDP-Gal (1 µCi) after 1 h incubation. Data from independent preparations of TgSkp1 are colored in different shades. FGaGn-Skp1 is included for comparison. Error bars represent S.D. of duplicate measurements. Inset shows Western blots of the Skp1 preparations used, where FGaGn-Skp1, which is recognized specifically by pAb UOK104, is largely converted in a 3.5-h reaction using Glt1 and UDP-Glc to GlFGaGn-Skp1, which is recognized only by the pan-specific pAb UOK75. ( I ) Reactions with synthetic oligosaccharides conjugated to pNP were conducted in parallel using the same conditions. ( J ) Biochemical complementation to detect Gat1 substrates. Desalted S100 extracts of RH and gat1 Δ/RH were reacted with recombinant Gat1 in the presence of UDP-[ 3 H]Gal, and the product of the reaction was separated on an SDS-PAGE gel which was sliced into 40 bands for liquid scintillation counting. The migration position of Skp1 is marked with an arrow. See Figs. S9H and S9I for trials using different strains.
    Figure Legend Snippet: Substrate specificity of Gat1. ( A ) Recombinantly expressed and purified preparations of TgGat1 and PuGat1 were analyzed by SDS-PAGE and staining with Coomassie blue. ( B ) Temporal dependence of UDP-Gal and UDP-Glc hydrolysis. The averages and standard deviations of 3 technical replicates are shown. A similar profile was observed with a different enzyme concentration. See Fig. S9E for a trial with higher enzyme concentrations. ( C ) Transferase activity utilizing 8 µM UDP-Gal or UDP-Glc toward 20 mM maltose-pNP for TgGat1 and PuGat1. The averages and standard deviations of two technical replicates are shown; similar profiles were in 2 independent assays with a different TgGat1 preparation. ( D ) UDP-Gal and UDP-Glc concentration dependence of TgGat1 transferase activity toward 20 mM maltose-pNP. The averages and standard deviations of two technical replicates are shown, and an independent trial with TgGat1 and PuGat1 against UDP-Gal is shown in Fig S9F. ( E ) Maltose-pNP concentration dependence of TgGat1 and PuGat1 transferase activity from 20 µM UDP-Gal. The averages and standard deviations of two technical replicates are shown. ( F ) Relative Gal-transferase activity of TgGat1 and PuGat1 toward different acceptors. The averages and standard deviations of three technical replicates are shown. Similar results were obtained in three independent trials. ( G ) Effect of UDP-Glc concentration on the Gal-transferase activity of TgGat1. Reactions were incubated for 1 h. The averages and standard deviations of two technical replicates are shown. (H ) Gal-transferase activity of TgGaT1 toward varied concentrations of GlFGaGn-Skp1, in the presence of 40 µM UDP-Gal (1 µCi) after 1 h incubation. Data from independent preparations of TgSkp1 are colored in different shades. FGaGn-Skp1 is included for comparison. Error bars represent S.D. of duplicate measurements. Inset shows Western blots of the Skp1 preparations used, where FGaGn-Skp1, which is recognized specifically by pAb UOK104, is largely converted in a 3.5-h reaction using Glt1 and UDP-Glc to GlFGaGn-Skp1, which is recognized only by the pan-specific pAb UOK75. ( I ) Reactions with synthetic oligosaccharides conjugated to pNP were conducted in parallel using the same conditions. ( J ) Biochemical complementation to detect Gat1 substrates. Desalted S100 extracts of RH and gat1 Δ/RH were reacted with recombinant Gat1 in the presence of UDP-[ 3 H]Gal, and the product of the reaction was separated on an SDS-PAGE gel which was sliced into 40 bands for liquid scintillation counting. The migration position of Skp1 is marked with an arrow. See Figs. S9H and S9I for trials using different strains.

    Techniques Used: Purification, SDS Page, Staining, Concentration Assay, Activity Assay, Incubation, Western Blot, Recombinant, Migration

    5) Product Images from "Conformational Epitope-Specific Broadly Neutralizing Plasma Antibodies Obtained from an HIV-1 Clade C-Infected Elite Neutralizer Mediate Autologous Virus Escape through Mutations in the V1 Loop"

    Article Title: Conformational Epitope-Specific Broadly Neutralizing Plasma Antibodies Obtained from an HIV-1 Clade C-Infected Elite Neutralizer Mediate Autologous Virus Escape through Mutations in the V1 Loop

    Journal: Journal of Virology

    doi: 10.1128/JVI.03090-15

    BCN plasma IgG to 4-2.J41 monomeric gp120 (A) and BG505-SOSIP.664-D7324 cleaved trimeric gp140 (B) soluble proteins was assessed by ELISA. IgG purified from HIV-negative
    Figure Legend Snippet: BCN plasma IgG to 4-2.J41 monomeric gp120 (A) and BG505-SOSIP.664-D7324 cleaved trimeric gp140 (B) soluble proteins was assessed by ELISA. IgG purified from HIV-negative

    Techniques Used: Enzyme-linked Immunosorbent Assay, Purification

    6) Product Images from "Analysis of Ribosome-Associated mRNAs in Rice Reveals the Importance of Transcript Size and GC Content in Translation"

    Article Title: Analysis of Ribosome-Associated mRNAs in Rice Reveals the Importance of Transcript Size and GC Content in Translation

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.116.036020

    Epitope-tagged rice RPL18 assembles into functional ribosomes that can be purified by TRAP. (A) Confirmation of RPL18 assembly into ribosomes. The p35S:HF-OsRPL18 rice line was used as the source of ribosomal complexes, which were separated by ultracentrifugation on a 20–60% (w/v) sucrose density gradient. The absorbance at 254 nm was recorded to detect the ribosomal subunits of 40S and 60S, monosomes (80S), and polysomes. The gradient was fractionated and proteins in the 10 fractions were analyzed by SDS-PAGE separation and western blotting processed with anti-FLAG (α-FLAG) or anti-RPS6 (α-RPS6) antisera. Molecular mass markers are indicated on the left. (B) Purification of polysomes by TRAP. Equal weights of pulverized tissue from untransformed Nipponbare (control) and homozygous transgenic p35S:HF-OsRPL18 shoots were solubilized in polysome extraction buffer to obtain a clarified supernatant (total). The extract was incubated with anti-FLAG-bound Dynabeads coupled to Protein G to bind HF-RPL18. The supernatant (unbound fraction) was collected to evaluate the efficiency of the immunopurification. The magnetically captured protein–RNA complexes (TRAP fraction) was eluted from the beads using 3X-FLAG peptide. Each fraction was analyzed by western blot with α-FLAG and α-RPS6. The expected molecular mass of HF-RPL18 is 25 kDa. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TRAP, translating ribosome affinity purification.
    Figure Legend Snippet: Epitope-tagged rice RPL18 assembles into functional ribosomes that can be purified by TRAP. (A) Confirmation of RPL18 assembly into ribosomes. The p35S:HF-OsRPL18 rice line was used as the source of ribosomal complexes, which were separated by ultracentrifugation on a 20–60% (w/v) sucrose density gradient. The absorbance at 254 nm was recorded to detect the ribosomal subunits of 40S and 60S, monosomes (80S), and polysomes. The gradient was fractionated and proteins in the 10 fractions were analyzed by SDS-PAGE separation and western blotting processed with anti-FLAG (α-FLAG) or anti-RPS6 (α-RPS6) antisera. Molecular mass markers are indicated on the left. (B) Purification of polysomes by TRAP. Equal weights of pulverized tissue from untransformed Nipponbare (control) and homozygous transgenic p35S:HF-OsRPL18 shoots were solubilized in polysome extraction buffer to obtain a clarified supernatant (total). The extract was incubated with anti-FLAG-bound Dynabeads coupled to Protein G to bind HF-RPL18. The supernatant (unbound fraction) was collected to evaluate the efficiency of the immunopurification. The magnetically captured protein–RNA complexes (TRAP fraction) was eluted from the beads using 3X-FLAG peptide. Each fraction was analyzed by western blot with α-FLAG and α-RPS6. The expected molecular mass of HF-RPL18 is 25 kDa. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TRAP, translating ribosome affinity purification.

    Techniques Used: Functional Assay, Purification, SDS Page, Western Blot, Transgenic Assay, Incubation, Immu-Puri, Polyacrylamide Gel Electrophoresis, Affinity Purification

    7) Product Images from "The long noncoding RNA lnc-EGFR stimulates T-regulatory cells differentiation thus promoting hepatocellular carcinoma immune evasion"

    Article Title: The long noncoding RNA lnc-EGFR stimulates T-regulatory cells differentiation thus promoting hepatocellular carcinoma immune evasion

    Journal: Nature Communications

    doi: 10.1038/ncomms15129

    Lnc-EGFR prevents the ubiquitination of EGFR by binding to Tyr1045. ( a ) T cells isolated from peripheral blood of HCC patients were transduced with indicated lentiviral particles and then treated with with EGF (20 ng ml −1 ) for indicated timepoints followed by western blotting for p-EGFR(Y1045), p-EGFR(Y1068), p-EGFR(Y1073), p-ERK1/2(T202/Y204), EGFR, ERK1/2 and β-actin. ( b ) Normal, healthy human T cells transduced with mock or indicated lentiviral particles were determined with real-time PCR (upper) and were further treated with EGF (100 ng ml −1 ) for 90 min or left untreated. Whole-cell lysates were prepared and EGFR was immunoprecipitated followed by western blotting for ubiquitin. Equal loading of EGFR was determined by western blotting via anti-EGFR antibodies (lower). ( c ) Whole-cells lysates were prepared and c-CBL was immunoprecipitated via anti-c-CBL antibody. The presence of EGFR in the immunecomplex was determined by western blotting via anti-EGFR antibody. ( d ) Transduced T cells were treated with anti-CD3/anti-CD28 beads (bead-to-cell ratio of 1:1), EGF (20 ng ml −1 ) or left untreated in the presence or absence of PD98059 (40 μM) and/or CsA (1 μM). Whole-cell lysates were prepared and subjected to Western blotting for p-ERK1/2(T202/Y204), p-MEK1/2(S217/221), p-NF-AT1(S54), ERK1/2, IL-2, MEK1/2 and β-actin. Each experiment was performed triplicated.
    Figure Legend Snippet: Lnc-EGFR prevents the ubiquitination of EGFR by binding to Tyr1045. ( a ) T cells isolated from peripheral blood of HCC patients were transduced with indicated lentiviral particles and then treated with with EGF (20 ng ml −1 ) for indicated timepoints followed by western blotting for p-EGFR(Y1045), p-EGFR(Y1068), p-EGFR(Y1073), p-ERK1/2(T202/Y204), EGFR, ERK1/2 and β-actin. ( b ) Normal, healthy human T cells transduced with mock or indicated lentiviral particles were determined with real-time PCR (upper) and were further treated with EGF (100 ng ml −1 ) for 90 min or left untreated. Whole-cell lysates were prepared and EGFR was immunoprecipitated followed by western blotting for ubiquitin. Equal loading of EGFR was determined by western blotting via anti-EGFR antibodies (lower). ( c ) Whole-cells lysates were prepared and c-CBL was immunoprecipitated via anti-c-CBL antibody. The presence of EGFR in the immunecomplex was determined by western blotting via anti-EGFR antibody. ( d ) Transduced T cells were treated with anti-CD3/anti-CD28 beads (bead-to-cell ratio of 1:1), EGF (20 ng ml −1 ) or left untreated in the presence or absence of PD98059 (40 μM) and/or CsA (1 μM). Whole-cell lysates were prepared and subjected to Western blotting for p-ERK1/2(T202/Y204), p-MEK1/2(S217/221), p-NF-AT1(S54), ERK1/2, IL-2, MEK1/2 and β-actin. Each experiment was performed triplicated.

    Techniques Used: Binding Assay, Isolation, Transduction, Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation

    8) Product Images from "A Subset of CXCR5+CD8+ T Cells in the Germinal Centers From Human Tonsils and Lymph Nodes Help B Cells Produce Immunoglobulins"

    Article Title: A Subset of CXCR5+CD8+ T Cells in the Germinal Centers From Human Tonsils and Lymph Nodes Help B Cells Produce Immunoglobulins

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02287

    CXCR5 + CD8 + T Cells provide help to B cells for the production of immunoglobulins. Physical contact between CD8 + T Cells (red) and B cells (green) in tonsil sections, and scale bars, 50μm. (A , n = 5). Sorted tonsil B cells and sorted CD8 + T Cells at the ratio of 1:1, 1:5, and 1:10 were co-cultured with or without α-CD3/CD28 dynabeads for 10 days (B,C) . Sorted B cells were co-cultured with fresh CD8 + or fixed CD8 + T Cells, fresh CD4 + or fixed CD4 + T Cells at the ratio of 5:1 in the presence of α-CD3/CD28 dynabeads for 10 days (D) . Sorted B cells and CD8 + , CXCR5 + CD8 + , CXCR5 − CD8 + T Cells at the ratio of 5:1 were co-cultured with or without anti-IL-21 and anti-CD40L in the presence of α-CD3/CD28 dynabeads for 10 days (E) . The supernatants from the different co-cultures were analyzed by ELISA for the production of IgG, IgM, and IgA. Data are expressed as the mean ± SD, and compared with Mann–Whitney test. * P
    Figure Legend Snippet: CXCR5 + CD8 + T Cells provide help to B cells for the production of immunoglobulins. Physical contact between CD8 + T Cells (red) and B cells (green) in tonsil sections, and scale bars, 50μm. (A , n = 5). Sorted tonsil B cells and sorted CD8 + T Cells at the ratio of 1:1, 1:5, and 1:10 were co-cultured with or without α-CD3/CD28 dynabeads for 10 days (B,C) . Sorted B cells were co-cultured with fresh CD8 + or fixed CD8 + T Cells, fresh CD4 + or fixed CD4 + T Cells at the ratio of 5:1 in the presence of α-CD3/CD28 dynabeads for 10 days (D) . Sorted B cells and CD8 + , CXCR5 + CD8 + , CXCR5 − CD8 + T Cells at the ratio of 5:1 were co-cultured with or without anti-IL-21 and anti-CD40L in the presence of α-CD3/CD28 dynabeads for 10 days (E) . The supernatants from the different co-cultures were analyzed by ELISA for the production of IgG, IgM, and IgA. Data are expressed as the mean ± SD, and compared with Mann–Whitney test. * P

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Expression of co-stimulated molecules on CXCR5 + CD8 + memory T cells from tonsils, lymph nodes and PBMCs. Upon stimulation with CD3/CD28 dynabeads for 8 h, the expression of CD40L and ICOS on CXCR5 + and CXCR5 − CD8 + memory T cells from tonsils, lymph nodes and PBMCs was detected by flow cytometry (A,B) . The data are representative of thirteen or fifteen independent experiments, and were analyzed by two-tailed unpaired t -test (C) . Error bar denote s.e.m. * P
    Figure Legend Snippet: Expression of co-stimulated molecules on CXCR5 + CD8 + memory T cells from tonsils, lymph nodes and PBMCs. Upon stimulation with CD3/CD28 dynabeads for 8 h, the expression of CD40L and ICOS on CXCR5 + and CXCR5 − CD8 + memory T cells from tonsils, lymph nodes and PBMCs was detected by flow cytometry (A,B) . The data are representative of thirteen or fifteen independent experiments, and were analyzed by two-tailed unpaired t -test (C) . Error bar denote s.e.m. * P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Two Tailed Test

    9) Product Images from "Immune Derangements in Patients with Myelofibrosis: The Role of Treg, Th17, and sIL2Rα"

    Article Title: Immune Derangements in Patients with Myelofibrosis: The Role of Treg, Th17, and sIL2Rα

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116723

    Function of regulatory T cell in MF patients. Treg function was measured as the percentage of suppression of cell proliferation of CD4 + CD25 - by CD4 + CD25 + cells using an XTT-based colorimetric assay. CD4 + CD25 - cells were cultured with CD4 + CD25 + cells, Dynabeads Human T Cell Activator CD3CD28 (Invitrogen) were added for 7 days, XTT-labeled reagent was added and incubated for 4 h at 37°C, 6.5% CO 2 , and spectrophotometric absorbance was then measured at 450 nm. The values of suppression are expressed as percentage of the values of suppression of proliferation response using CD4 + CD25 - T cells cultured alone in the absence of CD4 + CD25 + T cells and were used as 100% of nonsuppression control. MF = myelofibrosis, MPN = myeloproliferative neoplasm, CTR = control.
    Figure Legend Snippet: Function of regulatory T cell in MF patients. Treg function was measured as the percentage of suppression of cell proliferation of CD4 + CD25 - by CD4 + CD25 + cells using an XTT-based colorimetric assay. CD4 + CD25 - cells were cultured with CD4 + CD25 + cells, Dynabeads Human T Cell Activator CD3CD28 (Invitrogen) were added for 7 days, XTT-labeled reagent was added and incubated for 4 h at 37°C, 6.5% CO 2 , and spectrophotometric absorbance was then measured at 450 nm. The values of suppression are expressed as percentage of the values of suppression of proliferation response using CD4 + CD25 - T cells cultured alone in the absence of CD4 + CD25 + T cells and were used as 100% of nonsuppression control. MF = myelofibrosis, MPN = myeloproliferative neoplasm, CTR = control.

    Techniques Used: Colorimetric Assay, Cell Culture, Labeling, Incubation

    10) Product Images from "Chimeric Antigen Receptors Based on Low Affinity Mutants of FcεRI Re-direct T Cell Specificity to Cells Expressing Membrane IgE"

    Article Title: Chimeric Antigen Receptors Based on Low Affinity Mutants of FcεRI Re-direct T Cell Specificity to Cells Expressing Membrane IgE

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02231

    M2 CAR mediates potent primary human T cell responses to U266 cells. (A) Purified human CD8 + T cells were activated using anti-CD3/anti-CD28 beads and transduced with lentiviral vectors encoding the M2 CAR (T-M2). Cells were stained with anti-FcεRIα-PE antibody and analyzed by flow cytometry 7 days post-transduction. Untransduced human CD8 + T cells (T) served as negative control. (B) Human CD8 + T cells transduced with the M2 CAR (T-M2) or untransduced control T cells (T) were stimulated with equal numbers of CD3/CD28 beads, U266 cells, or U266 cells in the presence of 10 μg/ml IgE for 20 h. The concentrations of IFNγ in the supernatant were determined using ELISA. (C) CD8 + T cells expressing the M2 CAR (T-M2) or control T cells (T) were cultured with U266 cells stably expressing firefly luciferase at indicated ratios for 20 h. The percentages of specific lysis were determined based on the luciferase activity in the remaining live U266-luci cells. Data are presented as mean ± SD ( n = 3). ** P
    Figure Legend Snippet: M2 CAR mediates potent primary human T cell responses to U266 cells. (A) Purified human CD8 + T cells were activated using anti-CD3/anti-CD28 beads and transduced with lentiviral vectors encoding the M2 CAR (T-M2). Cells were stained with anti-FcεRIα-PE antibody and analyzed by flow cytometry 7 days post-transduction. Untransduced human CD8 + T cells (T) served as negative control. (B) Human CD8 + T cells transduced with the M2 CAR (T-M2) or untransduced control T cells (T) were stimulated with equal numbers of CD3/CD28 beads, U266 cells, or U266 cells in the presence of 10 μg/ml IgE for 20 h. The concentrations of IFNγ in the supernatant were determined using ELISA. (C) CD8 + T cells expressing the M2 CAR (T-M2) or control T cells (T) were cultured with U266 cells stably expressing firefly luciferase at indicated ratios for 20 h. The percentages of specific lysis were determined based on the luciferase activity in the remaining live U266-luci cells. Data are presented as mean ± SD ( n = 3). ** P

    Techniques Used: Purification, Transduction, Staining, Flow Cytometry, Cytometry, Negative Control, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Stable Transfection, Luciferase, Lysis, Activity Assay

    11) Product Images from "TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia"

    Article Title: TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2017.1371399

    TIGIT blockade decreases CLL viability and interferes with production of prosurvival cytokines. (a, b) Impact of TIGIT blockade on cytokine production by CD4+ T cells. PBMCs (upper panel; n = 12) or purified T cells (bottom panel; n = 10) were activated with CD3/CD28 beads for 24 h in the presence of recombinant TIGIT-Fc protein (rhTIGIT Fc) or corresponding isotype control and cytokines were quantifiued by intracellular FACS staining. (b) FACS analysis of surface expression of TIGIT was performed on peripheral blood samples from CLL patients (n = 12). Plot of percentages of CD4+TIGIT+ T cells are shown, discriminating between TIGIT low (
    Figure Legend Snippet: TIGIT blockade decreases CLL viability and interferes with production of prosurvival cytokines. (a, b) Impact of TIGIT blockade on cytokine production by CD4+ T cells. PBMCs (upper panel; n = 12) or purified T cells (bottom panel; n = 10) were activated with CD3/CD28 beads for 24 h in the presence of recombinant TIGIT-Fc protein (rhTIGIT Fc) or corresponding isotype control and cytokines were quantifiued by intracellular FACS staining. (b) FACS analysis of surface expression of TIGIT was performed on peripheral blood samples from CLL patients (n = 12). Plot of percentages of CD4+TIGIT+ T cells are shown, discriminating between TIGIT low (

    Techniques Used: Purification, Recombinant, FACS, Staining, Expressing

    CD4+ TIGIT+ cells provide a supportive microenvironment for CLL cells. (a) Representative dot plots showing gating strategy for flow cytometric cell sorting. (b) PBMCs have been depleted of TIGIT+, PD-1+ or TIGIT+PD-1+ CD4+ or CD8+ cells followed by incubation with CD3/CD28 activating beads. After 5 days in culture, CLL viability was measured and corresponding T/ CLL ratios were analysed (n = 6) (c).
    Figure Legend Snippet: CD4+ TIGIT+ cells provide a supportive microenvironment for CLL cells. (a) Representative dot plots showing gating strategy for flow cytometric cell sorting. (b) PBMCs have been depleted of TIGIT+, PD-1+ or TIGIT+PD-1+ CD4+ or CD8+ cells followed by incubation with CD3/CD28 activating beads. After 5 days in culture, CLL viability was measured and corresponding T/ CLL ratios were analysed (n = 6) (c).

    Techniques Used: Flow Cytometry, FACS, Incubation

    TIGIT+ cells display a distinct cytokine profile. (a) Representative dot plots showing intracellular cytokine production after cultivating CLL PBMCs for 24 h with CD3/CD28 activating beads. (b) Cytokine production of TIGIT- or TIGIT+CD4+ T cells in 14 samples. (c) Mean fluorescence intensity ratio (MFIR) of CD155 and CD112 on CD5+CD19+ CLL (top) or CD5+ T cells (bottom). The histograms show representative FACS plots of CLL cells (gated for CD5+CD19+cells) and T cells (CD5+ cells) stained with isotype controls (in gray) and CD112/CD155 specific antibodies (in black). The dot plots show results from n = 14 samples.
    Figure Legend Snippet: TIGIT+ cells display a distinct cytokine profile. (a) Representative dot plots showing intracellular cytokine production after cultivating CLL PBMCs for 24 h with CD3/CD28 activating beads. (b) Cytokine production of TIGIT- or TIGIT+CD4+ T cells in 14 samples. (c) Mean fluorescence intensity ratio (MFIR) of CD155 and CD112 on CD5+CD19+ CLL (top) or CD5+ T cells (bottom). The histograms show representative FACS plots of CLL cells (gated for CD5+CD19+cells) and T cells (CD5+ cells) stained with isotype controls (in gray) and CD112/CD155 specific antibodies (in black). The dot plots show results from n = 14 samples.

    Techniques Used: Fluorescence, FACS, Staining

    12) Product Images from "Characterization of Liver Monocytic Myeloid-Derived Suppressor Cells and Their Role in a Murine Model of Non-Alcoholic Fatty Liver Disease"

    Article Title: Characterization of Liver Monocytic Myeloid-Derived Suppressor Cells and Their Role in a Murine Model of Non-Alcoholic Fatty Liver Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149948

    The suppressive function of liver SSC low CD11b + Gr1 dim cells is dependent on a NO mechanism. (A) Proliferation of carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labeled T cells cultured in the presence of Dynabeads mouse T-Activator CD3/CD28 along with purified liver SSC low CD11b + Gr1 dim cells. Different enzyme inhibitors (L-NIL, nor-NOHA, or catalase) were added at the start of each respective culture. (B) Nitrite levels were investigated in the culture supernatants after 60 h of co-culture (n = 3). (C) The intracellular iNOS expression was determined by flow cytometry. The percentage of positive cells is indicated. The data obtained from 3 separate experiments are shown. * P
    Figure Legend Snippet: The suppressive function of liver SSC low CD11b + Gr1 dim cells is dependent on a NO mechanism. (A) Proliferation of carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labeled T cells cultured in the presence of Dynabeads mouse T-Activator CD3/CD28 along with purified liver SSC low CD11b + Gr1 dim cells. Different enzyme inhibitors (L-NIL, nor-NOHA, or catalase) were added at the start of each respective culture. (B) Nitrite levels were investigated in the culture supernatants after 60 h of co-culture (n = 3). (C) The intracellular iNOS expression was determined by flow cytometry. The percentage of positive cells is indicated. The data obtained from 3 separate experiments are shown. * P

    Techniques Used: Labeling, Cell Culture, Purification, Co-Culture Assay, Expressing, Flow Cytometry, Cytometry

    SSC low CD11b + Gr1 dim cells in the livers suppress T cell responses. (A) Proliferation of CFSE-labeled T cells cultured in the presence of Dynabeads mouse T-Activator CD3/CD28 with or without liver SSC high or SSC low CD11b + Gr1 dim cells. (B) Representative image of intracellular interferon-γ staining for T cells cultured with or without liver SSC high or SSC low CD11b + Gr1 dim cells. (C) T cells and allogenic dendritic cells were co-cultured. Liver SSC high and SSC low CD11b + Gr1 dim cells were added to the cultures. The data obtained from 3 separate experiments are shown. * P
    Figure Legend Snippet: SSC low CD11b + Gr1 dim cells in the livers suppress T cell responses. (A) Proliferation of CFSE-labeled T cells cultured in the presence of Dynabeads mouse T-Activator CD3/CD28 with or without liver SSC high or SSC low CD11b + Gr1 dim cells. (B) Representative image of intracellular interferon-γ staining for T cells cultured with or without liver SSC high or SSC low CD11b + Gr1 dim cells. (C) T cells and allogenic dendritic cells were co-cultured. Liver SSC high and SSC low CD11b + Gr1 dim cells were added to the cultures. The data obtained from 3 separate experiments are shown. * P

    Techniques Used: Labeling, Cell Culture, Staining

    13) Product Images from "High salt diet stimulates gut Th17 response and exacerbates TNBS-induced colitis in mice"

    Article Title: High salt diet stimulates gut Th17 response and exacerbates TNBS-induced colitis in mice

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13783

    HSD does not alter T helper 1 (Th1) response but impairs the Th17:Treg balance A. and B. IL-17A-GFP mice were exposed to 3 weeks of ND or HSD before sacrifice, the proportion of IFN-γ + and GFP IL-17A + cells within the CD4 + TCRβ + SI LP lymphocytes were analyzed ( n = 6), data were acquired by intracellular staining from the SI LP lymphocytes which were incubated for 4 hr with PMA/Ionomycin and GolgiPlug. C. - G. Measurements of the SI LP cells that isolated from the mice on a ND or HSD diet for 3 weeks. C, the flow cytometric gating strategy for CD4 + CD25 + Foxp3 + Treg cells, the Foxp3 level was analyzed after gated on CD4 + CD25 + cells, in the right panel, dash line represent the Foxp3 isotype control and the solid line represent the Foxp3 staining. D, the summative data of the Foxp3 expression and the percentages of CD4 + CD25 + Foxp3 + Treg cells in the SI LP cells ( n = 8). E-F, the IL-10 production of the CD4 + CD25 + Foxp3 + cells and the CD4 + Foxp3 − cells, fresh isolated SI LP cells were incubated for 4 hr with PMA/Ionomycin and GolgiPlug before intracellular staining by True-Nuclear TM Transcription Factor Buffer Set. G, the RORγt level of the SI LP CD4 + TCRβ + cells ( n = 6). H-I, Treg suppression assay, the CD4 effector cells (CD4 + CD62L + CD44 − ) were sorted from mice exposed to 3 weeks of ND, the CD4 + CD25 + Treg were sorted from mice exposed to 3 weeks of ND or HSD, CD4 effector cells were labeled with CFSE, stimulated with CD3/CD28 Dynabeads and cultured alone or co-cultured with Treg at ratios as indicated (with IL-2). H. CFSE dilution of CD4 effector cells was measured by flow cytometry after 3.5 days, and CFSE dilution was obtained after gating on CD62L + cells (CD4 effector cells). I. Summary of the proliferation rate of the CD4 effector cells. Data are expressed as mean ± SEM from three independent experiments.
    Figure Legend Snippet: HSD does not alter T helper 1 (Th1) response but impairs the Th17:Treg balance A. and B. IL-17A-GFP mice were exposed to 3 weeks of ND or HSD before sacrifice, the proportion of IFN-γ + and GFP IL-17A + cells within the CD4 + TCRβ + SI LP lymphocytes were analyzed ( n = 6), data were acquired by intracellular staining from the SI LP lymphocytes which were incubated for 4 hr with PMA/Ionomycin and GolgiPlug. C. - G. Measurements of the SI LP cells that isolated from the mice on a ND or HSD diet for 3 weeks. C, the flow cytometric gating strategy for CD4 + CD25 + Foxp3 + Treg cells, the Foxp3 level was analyzed after gated on CD4 + CD25 + cells, in the right panel, dash line represent the Foxp3 isotype control and the solid line represent the Foxp3 staining. D, the summative data of the Foxp3 expression and the percentages of CD4 + CD25 + Foxp3 + Treg cells in the SI LP cells ( n = 8). E-F, the IL-10 production of the CD4 + CD25 + Foxp3 + cells and the CD4 + Foxp3 − cells, fresh isolated SI LP cells were incubated for 4 hr with PMA/Ionomycin and GolgiPlug before intracellular staining by True-Nuclear TM Transcription Factor Buffer Set. G, the RORγt level of the SI LP CD4 + TCRβ + cells ( n = 6). H-I, Treg suppression assay, the CD4 effector cells (CD4 + CD62L + CD44 − ) were sorted from mice exposed to 3 weeks of ND, the CD4 + CD25 + Treg were sorted from mice exposed to 3 weeks of ND or HSD, CD4 effector cells were labeled with CFSE, stimulated with CD3/CD28 Dynabeads and cultured alone or co-cultured with Treg at ratios as indicated (with IL-2). H. CFSE dilution of CD4 effector cells was measured by flow cytometry after 3.5 days, and CFSE dilution was obtained after gating on CD62L + cells (CD4 effector cells). I. Summary of the proliferation rate of the CD4 effector cells. Data are expressed as mean ± SEM from three independent experiments.

    Techniques Used: Mouse Assay, Staining, Incubation, Isolation, Flow Cytometry, Expressing, Suppression Assay, Labeling, Cell Culture, Cytometry

    14) Product Images from "MAPKAP kinase 3 suppresses Ifng gene expression and attenuates NK cell cytotoxicity and Th1 CD4 T-cell development upon influenza A virus infection"

    Article Title: MAPKAP kinase 3 suppresses Ifng gene expression and attenuates NK cell cytotoxicity and Th1 CD4 T-cell development upon influenza A virus infection

    Journal: The FASEB Journal

    doi: 10.1096/fj.14-249599

    CD4 + T cells from Mk3 −/− mice respond with a stronger induction of IFN-γ in comparison to WT mice. A , B ) Splenic CD4 + T cells from C57Bl/6 WT and Mk3 −/− mice were isolated and activated with anti-CD3+anti-CD28 Dynabeads (Life Technologies) for 5 d, after which they were stimulated with IL-2 (neutral), IL-2 + IL-12 (Th1), or IL-2 + IL-4 (Th2) for a further 2 d. Supernatants were then analyzed for secreted IFN-γ ( A ), and the cells for intracellular expression of synthesized IFN-γ ( B ). Mean intracellular IFN-γ level of WT cells cultured under neutral conditions was assigned a value of 1 ( B ). * P
    Figure Legend Snippet: CD4 + T cells from Mk3 −/− mice respond with a stronger induction of IFN-γ in comparison to WT mice. A , B ) Splenic CD4 + T cells from C57Bl/6 WT and Mk3 −/− mice were isolated and activated with anti-CD3+anti-CD28 Dynabeads (Life Technologies) for 5 d, after which they were stimulated with IL-2 (neutral), IL-2 + IL-12 (Th1), or IL-2 + IL-4 (Th2) for a further 2 d. Supernatants were then analyzed for secreted IFN-γ ( A ), and the cells for intracellular expression of synthesized IFN-γ ( B ). Mean intracellular IFN-γ level of WT cells cultured under neutral conditions was assigned a value of 1 ( B ). * P

    Techniques Used: Mouse Assay, Isolation, Expressing, Synthesized, Cell Culture

    15) Product Images from "A Full GMP Process to Select and Amplify Epitope-Specific T Lymphocytes for Adoptive Immunotherapy of Metastatic Melanoma"

    Article Title: A Full GMP Process to Select and Amplify Epitope-Specific T Lymphocytes for Adoptive Immunotherapy of Metastatic Melanoma

    Journal: Clinical and Developmental Immunology

    doi: 10.1155/2013/932318

    Sorting procedure. (a) 10 7 Chim-AvT dynabeads are coated with HLA-A2-peptide monomers, and coating efficiency is assessed on 10 5 beads, by labelling with an PE-labelled anti-HLA-A2 mAb (5 μ g/mL). (b) Peptide stimulated T cells, containing at least 1% of specific T cells are incubated with Clinimers (ratio 1/1) and recovered on a magnet. After washes in PBS, rosetted T cells are seeded in 96-well plates (10 3 cells/well), containing irradiated allogeneic feeder cells, for amplification. After 14 days, the specificity of T cells is assessed by double labelling with an anti-CD8 mAb and each specific tetramer. (c) Influence of the number of washes on sorting yields. Sorting yields are calculated by dividing the number of rosetted T lymphocytes counted after the sort by the number of specific T cells in the sorted population estimated by tetramer staining. Blue bars represent Melan-A-specific T cells and red bars MELOE-1-specific ones. (d) Influence of the number of washes on purity of selected and amplified T cells. The purity of amplified sorted-T cells is assessed after the 14-day amplification period on feeder cells, by double staining with an anti-CD8 mAb and the specific tetramer. Blue bars represent Melan-A-specific T cells and red bars MELOE-1-specific ones.
    Figure Legend Snippet: Sorting procedure. (a) 10 7 Chim-AvT dynabeads are coated with HLA-A2-peptide monomers, and coating efficiency is assessed on 10 5 beads, by labelling with an PE-labelled anti-HLA-A2 mAb (5 μ g/mL). (b) Peptide stimulated T cells, containing at least 1% of specific T cells are incubated with Clinimers (ratio 1/1) and recovered on a magnet. After washes in PBS, rosetted T cells are seeded in 96-well plates (10 3 cells/well), containing irradiated allogeneic feeder cells, for amplification. After 14 days, the specificity of T cells is assessed by double labelling with an anti-CD8 mAb and each specific tetramer. (c) Influence of the number of washes on sorting yields. Sorting yields are calculated by dividing the number of rosetted T lymphocytes counted after the sort by the number of specific T cells in the sorted population estimated by tetramer staining. Blue bars represent Melan-A-specific T cells and red bars MELOE-1-specific ones. (d) Influence of the number of washes on purity of selected and amplified T cells. The purity of amplified sorted-T cells is assessed after the 14-day amplification period on feeder cells, by double staining with an anti-CD8 mAb and the specific tetramer. Blue bars represent Melan-A-specific T cells and red bars MELOE-1-specific ones.

    Techniques Used: Incubation, Irradiation, Amplification, Staining, Double Staining

    16) Product Images from "Virome Capture Sequencing Enables Sensitive Viral Diagnosis and Comprehensive Virome Analysis"

    Article Title: Virome Capture Sequencing Enables Sensitive Viral Diagnosis and Comprehensive Virome Analysis

    Journal: mBio

    doi: 10.1128/mBio.01491-15

    VirCapSeq-VERT enhances the performance of high-throughput sequencing by increasing the number of mapped viral reads recovered from high-background specimens. Eight different viral NAs were quantitated by qPCR and used to spike a background of lung-derived (3 viruses) or blood-derived (5 viruses) NA extracts. Samples were split in two and processed by standard HTS (blue) or with VirCapSeq-VERT (red). FLUAV, influenza A virus; EVD-68, enterovirus D68; MERS-CoV, MERS coronavirus; DENV, dengue virus; EBOV, Ebola virus; WNV, West Nile virus; CVV, Cache Valley virus; HHV-1, human herpesvirus 1.
    Figure Legend Snippet: VirCapSeq-VERT enhances the performance of high-throughput sequencing by increasing the number of mapped viral reads recovered from high-background specimens. Eight different viral NAs were quantitated by qPCR and used to spike a background of lung-derived (3 viruses) or blood-derived (5 viruses) NA extracts. Samples were split in two and processed by standard HTS (blue) or with VirCapSeq-VERT (red). FLUAV, influenza A virus; EVD-68, enterovirus D68; MERS-CoV, MERS coronavirus; DENV, dengue virus; EBOV, Ebola virus; WNV, West Nile virus; CVV, Cache Valley virus; HHV-1, human herpesvirus 1.

    Techniques Used: Next-Generation Sequencing, Real-time Polymerase Chain Reaction, Derivative Assay

    Read coverage versus probe coverage of VirCapSeq-VERT for West Nile virus (A), Cache Valley virus (B), and MERS coronavirus (C). Virus genomes are represented by horizontal black lines and coding sequence by black pointed boxes. The top graph in each panel indicates the read coverage obtained by VirCapSeq-VERT; probe coverage is shown below. Colored lines indicate mismatch to the reference used for alignment (green, A; red, T; blue, C; orange, G). Line heights indicate the frequency of the mismatched bases.
    Figure Legend Snippet: Read coverage versus probe coverage of VirCapSeq-VERT for West Nile virus (A), Cache Valley virus (B), and MERS coronavirus (C). Virus genomes are represented by horizontal black lines and coding sequence by black pointed boxes. The top graph in each panel indicates the read coverage obtained by VirCapSeq-VERT; probe coverage is shown below. Colored lines indicate mismatch to the reference used for alignment (green, A; red, T; blue, C; orange, G). Line heights indicate the frequency of the mismatched bases.

    Techniques Used: Sequencing

    Selective enhancement of vertebrate virus detection by VirCapSeq-VERT. Bat fecal sample material was divided in two and analyzed using HTS with filtration and nuclease digest combined with postextraction DNase treatment or using VirCapSeq-VERT alone. VirCapSeq-VERT reduced the number of nonvertebrate viral reads and efficiently sequenced vertebrate virus sequences detected only at low levels by conventional HTS.
    Figure Legend Snippet: Selective enhancement of vertebrate virus detection by VirCapSeq-VERT. Bat fecal sample material was divided in two and analyzed using HTS with filtration and nuclease digest combined with postextraction DNase treatment or using VirCapSeq-VERT alone. VirCapSeq-VERT reduced the number of nonvertebrate viral reads and efficiently sequenced vertebrate virus sequences detected only at low levels by conventional HTS.

    Techniques Used: Filtration

    Limit of detection for VirCapSeq-VERT. Total nucleic acid from blood or lung tissue was spiked with human herpesvirus 1 (HHV-1) and West Nile virus (WNV) nucleic acid. The two preparations were serially diluted to generate six samples containing both viruses at 5,000, 1,000, 300, 100, 30, or 10 copies in 100 ng lung tissue or 50 ng whole-blood nucleic acid. Samples were processed with VirCapSeq-VERT.
    Figure Legend Snippet: Limit of detection for VirCapSeq-VERT. Total nucleic acid from blood or lung tissue was spiked with human herpesvirus 1 (HHV-1) and West Nile virus (WNV) nucleic acid. The two preparations were serially diluted to generate six samples containing both viruses at 5,000, 1,000, 300, 100, 30, or 10 copies in 100 ng lung tissue or 50 ng whole-blood nucleic acid. Samples were processed with VirCapSeq-VERT.

    Techniques Used:

    Efficiency of viral read mapping with VirCapSeq-VERT. Human blood and serum were spiked with live enterovirus D68 virus stock quantitated by qPCR to generate samples with 500, 200, 100, or 10 copies/ml. Five hundred microliters of each sample was extracted and processed with VirCapSeq-VERT.
    Figure Legend Snippet: Efficiency of viral read mapping with VirCapSeq-VERT. Human blood and serum were spiked with live enterovirus D68 virus stock quantitated by qPCR to generate samples with 500, 200, 100, or 10 copies/ml. Five hundred microliters of each sample was extracted and processed with VirCapSeq-VERT.

    Techniques Used: Real-time Polymerase Chain Reaction

    In silico validation of the VirCapSeq-VERT probe design. Probe depth and coverage of the VirCapSeq-VERT probe library are shown for poliovirus (A), yellow fever virus (B), and parvovirus B19 (C). Virus genomes are represented by black lines. The coding sequences are represented by green boxes. The probes are indicated by grey boxes. The top graph in each panel indicates probe depth at each locus. Colored lines in the probes indicate mismatch to the reference used for alignment (green, A; red, T; blue, C; orange, G). Line heights in the coverage track above indicate frequency of the mismatched bases.
    Figure Legend Snippet: In silico validation of the VirCapSeq-VERT probe design. Probe depth and coverage of the VirCapSeq-VERT probe library are shown for poliovirus (A), yellow fever virus (B), and parvovirus B19 (C). Virus genomes are represented by black lines. The coding sequences are represented by green boxes. The probes are indicated by grey boxes. The top graph in each panel indicates probe depth at each locus. Colored lines in the probes indicate mismatch to the reference used for alignment (green, A; red, T; blue, C; orange, G). Line heights in the coverage track above indicate frequency of the mismatched bases.

    Techniques Used: In Silico

    17) Product Images from "Wnt/β-catenin signaling regulates VE-cadherin-mediated anastomosis of brain capillaries by counteracting S1pr1 signaling"

    Article Title: Wnt/β-catenin signaling regulates VE-cadherin-mediated anastomosis of brain capillaries by counteracting S1pr1 signaling

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07302-x

    Junctional localization of VE-cadherin and Esama is affected by Wnt signaling inhibition. a Schematic of the zebrafish hindbrain vasculature at 42 hpf. Red box indicates region of analysis. In the following images ( b – d ) half of the boxed region is displayed. b – d Inhibition of Wnt signaling by IWR-1 from 29 hpf ( b ) or by dnTcf expression ( c , d ) strongly reduced expression of VE-cadherin or Esama and ZO-1 at the cell–cell junctions. Immunostaining for VE-cadherin ( b , c ; red) or Esama ( b , d ; red) and ZO-1 ( b , c ; green). Single channel images were displayed in inverted colors for better visualization. b In control embryos (DMSO), VE-cadherin and Esama were detected in cell–cell junctions along the CtAs and in anastomosis rings (arrow). Inhibition of Wnt signaling resulted in reduced staining of VE-cadherin and Esama at the cell junctions, formation of multiple small anastomosis rings (arrows) and ectopic VE-cadherin-positive cell protrusions (asterisks). c , d In control embryos (mCherry iEC ), VE-cadherin and Esama are strongly expressed in cell–cell junctions. Expression of mCherry-dnTcf iEC dramatically reduced VE-cadherin and ZO-1 ( c ) or Esama ( d ) at the cell–cell junctions. e Primary mouse BECs enriched from microvascular fragments from P3 mice exhibited impaired VE-cadherin junction formation following Wnt signaling inhibition by IWR-1 (DMSO: n = 86; IWR: n = 80; N = 4). In contrast, BECs isolated from adult mice had coverage of cell–cell contact sides by VE-cadherin similar to control (DMSO: n = 143; IWR-1: n = 130; N = 4). Immunostaining for VE-cadherin (red), ZO-1 (green), CD31 (white), and DAPI (blue) of cultured primary mouse BECs after treatment with IWR-1 (P3: 10 µM; adult: 20 µM) or DMSO, respectively. Values represent mean ± SD. * p
    Figure Legend Snippet: Junctional localization of VE-cadherin and Esama is affected by Wnt signaling inhibition. a Schematic of the zebrafish hindbrain vasculature at 42 hpf. Red box indicates region of analysis. In the following images ( b – d ) half of the boxed region is displayed. b – d Inhibition of Wnt signaling by IWR-1 from 29 hpf ( b ) or by dnTcf expression ( c , d ) strongly reduced expression of VE-cadherin or Esama and ZO-1 at the cell–cell junctions. Immunostaining for VE-cadherin ( b , c ; red) or Esama ( b , d ; red) and ZO-1 ( b , c ; green). Single channel images were displayed in inverted colors for better visualization. b In control embryos (DMSO), VE-cadherin and Esama were detected in cell–cell junctions along the CtAs and in anastomosis rings (arrow). Inhibition of Wnt signaling resulted in reduced staining of VE-cadherin and Esama at the cell junctions, formation of multiple small anastomosis rings (arrows) and ectopic VE-cadherin-positive cell protrusions (asterisks). c , d In control embryos (mCherry iEC ), VE-cadherin and Esama are strongly expressed in cell–cell junctions. Expression of mCherry-dnTcf iEC dramatically reduced VE-cadherin and ZO-1 ( c ) or Esama ( d ) at the cell–cell junctions. e Primary mouse BECs enriched from microvascular fragments from P3 mice exhibited impaired VE-cadherin junction formation following Wnt signaling inhibition by IWR-1 (DMSO: n = 86; IWR: n = 80; N = 4). In contrast, BECs isolated from adult mice had coverage of cell–cell contact sides by VE-cadherin similar to control (DMSO: n = 143; IWR-1: n = 130; N = 4). Immunostaining for VE-cadherin (red), ZO-1 (green), CD31 (white), and DAPI (blue) of cultured primary mouse BECs after treatment with IWR-1 (P3: 10 µM; adult: 20 µM) or DMSO, respectively. Values represent mean ± SD. * p

    Techniques Used: Inhibition, Expressing, Immunostaining, Staining, Mouse Assay, Isolation, Cell Culture

    18) Product Images from "Discovery of Chromatin-Associated Proteins via Sequence-Specific Capture and Mass Spectrometric Protein Identification in Saccharomycescerevisiae"

    Article Title: Discovery of Chromatin-Associated Proteins via Sequence-Specific Capture and Mass Spectrometric Protein Identification in Saccharomycescerevisiae

    Journal: Journal of Proteome Research

    doi: 10.1021/pr5004938

    HyCCAPP is a multistep process that uses sequence-specific hybridization to capture DNA loci of interest from formaldehyde cross-linked cells. The overall procedure involves (1) cross-linking cells with formaldehyde followed by cell lysis, (2) hybridization capture from sonicated and RNaseA-treated lysate using desthiobiotin oligonucleotide capture probes and streptavidin-coated magnetic beads, (3) mass spectrometric analysis of captured proteins using LC–MS/MS, and (4) bioinformatic analysis of mass spectrometric data to determine the proteins enriched at the captured DNA locus of interest relative to non-enriched yeast lysate.
    Figure Legend Snippet: HyCCAPP is a multistep process that uses sequence-specific hybridization to capture DNA loci of interest from formaldehyde cross-linked cells. The overall procedure involves (1) cross-linking cells with formaldehyde followed by cell lysis, (2) hybridization capture from sonicated and RNaseA-treated lysate using desthiobiotin oligonucleotide capture probes and streptavidin-coated magnetic beads, (3) mass spectrometric analysis of captured proteins using LC–MS/MS, and (4) bioinformatic analysis of mass spectrometric data to determine the proteins enriched at the captured DNA locus of interest relative to non-enriched yeast lysate.

    Techniques Used: Sequencing, Hybridization, Lysis, Sonication, Magnetic Beads, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    19) Product Images from "Patch-Seq Protocol to Analyze the Electrophysiology, Morphology and Transcriptome of Whole Single Neurons Derived From Human Pluripotent Stem Cells"

    Article Title: Patch-Seq Protocol to Analyze the Electrophysiology, Morphology and Transcriptome of Whole Single Neurons Derived From Human Pluripotent Stem Cells

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00261

    Expression analysis of ERCC RNA spike-in controls to assess technical performance of SMARTer cDNA preparation. (A–C) Expression of a high-, medium-, and low-copy ERCC standard in n = 28 spiked cells measured by qRT-PCR and RNA-seq. Variability in expression increases with a decrease in absolute ERCC copy number. (D) Measured expression of ERCC RNA spike-ins as a function of the number of actual molecules added to each sample. The very small amount (∼10 pg) of RNA in a single cell requires a significant dilution of the ERCC stock to avoid overspiking of the experimental sample with exogenous transcripts. With a 1:5,000,000 dilution in the final master mix before reverse transcription, 73 ERCCs were present in at least one copy number, 71 of which were detected by RNA-seq across samples ( n = 28). (E) The variation in measured ERCC expression levels is highest for low expressed (i.e., low abundant) ERCCs.
    Figure Legend Snippet: Expression analysis of ERCC RNA spike-in controls to assess technical performance of SMARTer cDNA preparation. (A–C) Expression of a high-, medium-, and low-copy ERCC standard in n = 28 spiked cells measured by qRT-PCR and RNA-seq. Variability in expression increases with a decrease in absolute ERCC copy number. (D) Measured expression of ERCC RNA spike-ins as a function of the number of actual molecules added to each sample. The very small amount (∼10 pg) of RNA in a single cell requires a significant dilution of the ERCC stock to avoid overspiking of the experimental sample with exogenous transcripts. With a 1:5,000,000 dilution in the final master mix before reverse transcription, 73 ERCCs were present in at least one copy number, 71 of which were detected by RNA-seq across samples ( n = 28). (E) The variation in measured ERCC expression levels is highest for low expressed (i.e., low abundant) ERCCs.

    Techniques Used: Expressing, Quantitative RT-PCR, RNA Sequencing Assay

    Natural variation in expression of housekeeping genes in single neurons. TaqMan quantitative real-time PCR was performed on SMARTer cDNA generated from n = 56 single human neurons successfully isolated for Patch-seq to profile the expression levels of ACTB and GAPDH housekeeping genes. ACTB and GAPDH were readily expressed though at variable levels from cell to cell as revealed by both qRT-PCR (A) and RNA-sequencing (B) . (C) Single-neuron expression of ACTB and GAPDH housekeeping genes is highly correlated between qRT-PCR and RNA-sequencing measurements.
    Figure Legend Snippet: Natural variation in expression of housekeeping genes in single neurons. TaqMan quantitative real-time PCR was performed on SMARTer cDNA generated from n = 56 single human neurons successfully isolated for Patch-seq to profile the expression levels of ACTB and GAPDH housekeeping genes. ACTB and GAPDH were readily expressed though at variable levels from cell to cell as revealed by both qRT-PCR (A) and RNA-sequencing (B) . (C) Single-neuron expression of ACTB and GAPDH housekeeping genes is highly correlated between qRT-PCR and RNA-sequencing measurements.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Generated, Isolation, Quantitative RT-PCR, RNA Sequencing Assay

    20) Product Images from "Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification"

    Article Title: Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111437

    Phenotypic selection using SBP-ΔLNGFR. 293Ts were transiently transfected or lentivirally transduced with pHRSIN-SE-PGK-SBP-ΔLNGFR-W (encoding EGFP and SBP-ΔLNGFR; A) or pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR; B) and stained with streptavidin-APC plus/minus anti-HLA-A2-PE. Transfected/transduced cells are either GFP+/streptavidin-APC+ or HLA-A2-low/streptavidin-APC+ (dashed circles). Primary human CD4+ T cells were lentivirally transduced with the same constructs then selected using Dynabeads Biotin Binder. Purity of cells before (green or blue) and after (red) selection was assessed by GFP fluorescence (C) or staining with anti-HLA-A2-PE (D). Transduced cells are either GFP+ or HLA-A2-low (dashed boxes). Background staining of untransfected/unstransduced controls is shown (grey).
    Figure Legend Snippet: Phenotypic selection using SBP-ΔLNGFR. 293Ts were transiently transfected or lentivirally transduced with pHRSIN-SE-PGK-SBP-ΔLNGFR-W (encoding EGFP and SBP-ΔLNGFR; A) or pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR; B) and stained with streptavidin-APC plus/minus anti-HLA-A2-PE. Transfected/transduced cells are either GFP+/streptavidin-APC+ or HLA-A2-low/streptavidin-APC+ (dashed circles). Primary human CD4+ T cells were lentivirally transduced with the same constructs then selected using Dynabeads Biotin Binder. Purity of cells before (green or blue) and after (red) selection was assessed by GFP fluorescence (C) or staining with anti-HLA-A2-PE (D). Transduced cells are either GFP+ or HLA-A2-low (dashed boxes). Background staining of untransfected/unstransduced controls is shown (grey).

    Techniques Used: Selection, Transfection, Transduction, shRNA, Staining, Construct, Fluorescence

    Optimised Antibody Free Magnetic Cell Sorting of primary human CD4+ T cells. Primary human CD4+ T cells were lentivirally transduced with pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR under the PGK promoter) and either rested for 2 weeks (pale blue) or re-stimulated with CD3/CD28 Dynabeads 3 days prior to analysis (dark blue). Cells were co-stained with anti-HLA-A2-PE and anti-LNGFR-APC, and expression levels of SBP-ΔLNGFR compared in HLA-A2-low cells (A). Transduction with pHRSIN-SE-PGK-SBP-ΔLNGFR-W was then compared with pHRSIN-SE-P2A-SBP-ΔLNGFR-W (encoding GFP-P2A-SBP-ΔLNGFR under the SFFV promoter; B). Transduced cells are GFP+/LNGFR-APC+ (dashed circles). Background staining of untransfected/unstransduced controls is shown (grey). Finally, primary human CD4+ T cells were transduced with the optimised pHRSIREN-S-SBP-ΔLNGFR-W and pHRSIN-SE-P2A-SBP-ΔLNGFR-W lentivectors (C) encoding 2 different shRNAs and 2 different exogenous genes. Following selection with Dynabeads Biotin Binder, purity was assessed by staining with anti-LNGFR-PE (D). Each datapoint represents % LNGFR+ for a different construct (shRNA or exogenous gene) and means and SEMs are shown. Viability and functional activity of selected (expressing a control shRNA) and mock (unselected) cells were compared (E). Viability was measured 4 days after selection, and cells either rested or re-stimulated with CD3/CD28 Dynabeads. Resting and re-stimulated cells were stained with CD69-APC (day 2) and enumerated using CytoCount beads (days 1–3). CD69 expression by resting (grey) versus re-stimulated mock (pale blue) and selected (pink) cells is shown. Fold-increases in viable cell numbers following re-stimulation (proliferation) were calculated using day 1 as a baseline. Experiments were conducted in triplicate and means and SEMs are shown. cPPT – central polypurine tract; RRE – Rev response element; * – packaging signal; LTR – long terminal repeat; WPRE – Woodchuck Hepatitis Virus post-transcriptional regulatory element.
    Figure Legend Snippet: Optimised Antibody Free Magnetic Cell Sorting of primary human CD4+ T cells. Primary human CD4+ T cells were lentivirally transduced with pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR under the PGK promoter) and either rested for 2 weeks (pale blue) or re-stimulated with CD3/CD28 Dynabeads 3 days prior to analysis (dark blue). Cells were co-stained with anti-HLA-A2-PE and anti-LNGFR-APC, and expression levels of SBP-ΔLNGFR compared in HLA-A2-low cells (A). Transduction with pHRSIN-SE-PGK-SBP-ΔLNGFR-W was then compared with pHRSIN-SE-P2A-SBP-ΔLNGFR-W (encoding GFP-P2A-SBP-ΔLNGFR under the SFFV promoter; B). Transduced cells are GFP+/LNGFR-APC+ (dashed circles). Background staining of untransfected/unstransduced controls is shown (grey). Finally, primary human CD4+ T cells were transduced with the optimised pHRSIREN-S-SBP-ΔLNGFR-W and pHRSIN-SE-P2A-SBP-ΔLNGFR-W lentivectors (C) encoding 2 different shRNAs and 2 different exogenous genes. Following selection with Dynabeads Biotin Binder, purity was assessed by staining with anti-LNGFR-PE (D). Each datapoint represents % LNGFR+ for a different construct (shRNA or exogenous gene) and means and SEMs are shown. Viability and functional activity of selected (expressing a control shRNA) and mock (unselected) cells were compared (E). Viability was measured 4 days after selection, and cells either rested or re-stimulated with CD3/CD28 Dynabeads. Resting and re-stimulated cells were stained with CD69-APC (day 2) and enumerated using CytoCount beads (days 1–3). CD69 expression by resting (grey) versus re-stimulated mock (pale blue) and selected (pink) cells is shown. Fold-increases in viable cell numbers following re-stimulation (proliferation) were calculated using day 1 as a baseline. Experiments were conducted in triplicate and means and SEMs are shown. cPPT – central polypurine tract; RRE – Rev response element; * – packaging signal; LTR – long terminal repeat; WPRE – Woodchuck Hepatitis Virus post-transcriptional regulatory element.

    Techniques Used: FACS, Transduction, shRNA, Staining, Expressing, Selection, Construct, Functional Assay, Activity Assay

    21) Product Images from "HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33"

    Article Title: HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33

    Journal: Immunity

    doi: 10.1016/j.immuni.2017.09.015

    HpARI Binds Active Murine and Human IL-33 (A) Murine IL-33 western blot (non-reducing) of HpARI immunoprecipitation of mouse lung homogenates, using anti-c-Myc antibody, or MOPC isotype control (iso). (B) Human IL-33 western blot (non-reducing) of HpARI immunoprecipitation of human lung homogenates, as in (A). (C) Characterization of the interaction of mouse IL-33 (mIL-33) with HpARI by surface plasmon resonance (SPR - BIAcore T200). Reference corrected single kinetic titration SPR binding curves (black), and a globally fitted 1:1 kinetic binding model (grey). (D) Characterization by SPR of the interaction of human IL-33 (hIL-33) with HpARI, as in (C). (E) IL-33 levels (ELISA) in supernatants of freeze-thawed murine lung cells, incubated at 37°C for 0, 1, 2, or 4 hr, before addition of 1 μg/ml HpARI, and a further incubation for 1 hr at 37°C. (F) Untreated or oxidized recombinant murine IL-33 immunoprecipitated with HpARI as in (A). (G) Untreated or oxidized recombinant human IL-33 immunoprecipitated with HpARI as in (B). (H) Immunoprecipitation experiments repeated with recombinant murine IL-1α, and probed with anti-murine IL-1α. Arrows indicate specific IL-33 or IL-1α bands, and IL-33 reduced (“red”) or oxidized (“ox”) bands. All data are representative of at least two independent repeats. Error bars show SEM.
    Figure Legend Snippet: HpARI Binds Active Murine and Human IL-33 (A) Murine IL-33 western blot (non-reducing) of HpARI immunoprecipitation of mouse lung homogenates, using anti-c-Myc antibody, or MOPC isotype control (iso). (B) Human IL-33 western blot (non-reducing) of HpARI immunoprecipitation of human lung homogenates, as in (A). (C) Characterization of the interaction of mouse IL-33 (mIL-33) with HpARI by surface plasmon resonance (SPR - BIAcore T200). Reference corrected single kinetic titration SPR binding curves (black), and a globally fitted 1:1 kinetic binding model (grey). (D) Characterization by SPR of the interaction of human IL-33 (hIL-33) with HpARI, as in (C). (E) IL-33 levels (ELISA) in supernatants of freeze-thawed murine lung cells, incubated at 37°C for 0, 1, 2, or 4 hr, before addition of 1 μg/ml HpARI, and a further incubation for 1 hr at 37°C. (F) Untreated or oxidized recombinant murine IL-33 immunoprecipitated with HpARI as in (A). (G) Untreated or oxidized recombinant human IL-33 immunoprecipitated with HpARI as in (B). (H) Immunoprecipitation experiments repeated with recombinant murine IL-1α, and probed with anti-murine IL-1α. Arrows indicate specific IL-33 or IL-1α bands, and IL-33 reduced (“red”) or oxidized (“ox”) bands. All data are representative of at least two independent repeats. Error bars show SEM.

    Techniques Used: Western Blot, Immunoprecipitation, SPR Assay, Titration, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant

    22) Product Images from "Biphasic ROS production, p53 and BIK dictate the mode of cell death in response to DNA damage in colon cancer cells"

    Article Title: Biphasic ROS production, p53 and BIK dictate the mode of cell death in response to DNA damage in colon cancer cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0182809

    BIK mediates cisplatin-induced cell death in HCT-116 wt cellular spheroids, but not in HCT-116 p53 -/- spheroids. (A) HCT-116 wt and HCT-116 p53 -/- cells were transfected with BIK siRNA or scrambled siRNA for 24h. Cells were subsequently grown in 24-well 3D Algimatrix plates in the presence of RNAi duplexes. Microscopic evaluation of spheroids was done to verify that siRNA treatments did not interfere with the 3D growth of HCT-116 cells. (B) The efficiency of BIK depletion by RNA interference in HCT-116 wt and HCT-116 p53 -/- was determined by immunoblot analysis. Actin was probed as loading control. (C) Spheroids were treated with cisplatin (200 μM) for 48h and cell viability was assessed by using alamarBlue assay (mean±SEM, n = 3, *P
    Figure Legend Snippet: BIK mediates cisplatin-induced cell death in HCT-116 wt cellular spheroids, but not in HCT-116 p53 -/- spheroids. (A) HCT-116 wt and HCT-116 p53 -/- cells were transfected with BIK siRNA or scrambled siRNA for 24h. Cells were subsequently grown in 24-well 3D Algimatrix plates in the presence of RNAi duplexes. Microscopic evaluation of spheroids was done to verify that siRNA treatments did not interfere with the 3D growth of HCT-116 cells. (B) The efficiency of BIK depletion by RNA interference in HCT-116 wt and HCT-116 p53 -/- was determined by immunoblot analysis. Actin was probed as loading control. (C) Spheroids were treated with cisplatin (200 μM) for 48h and cell viability was assessed by using alamarBlue assay (mean±SEM, n = 3, *P

    Techniques Used: Transfection, Alamar Blue Assay

    23) Product Images from "Low oxygen post conditioning prevents thalamic secondary neuronal loss caused by excitotoxicity after cortical stroke"

    Article Title: Low oxygen post conditioning prevents thalamic secondary neuronal loss caused by excitotoxicity after cortical stroke

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39493-8

    NMDAR mediated nNOS activation is prevented by LOPC at 15 days. ( A) Calcium accumulation in the thalamus is not affected by LOPC at all pixel intensities as shown by cumulative tresholding analysis (scalebar = 400 μm). ( B ) The thalamic expression NR1, N2B and nNOS do not change in time or with treatment as assessed by WB. At 15 days, WB analysis show that the expression of synaptic marker PSD-95 is decreased by 50% in LOPC while Synapsin 1 expression is unchanged. ( C ) Co-immunoprecipitation of N2B and PSD-95 shows decreased interaction in LOPC samples between NMDAR and PSD-95 at 15 days (-Ab: beads not coated incubated in lysate; -Lysate: beads coated incubated in PBS). Images cropped from Supp. Fig. 3 . Results are shown as the mean ± SD. *p
    Figure Legend Snippet: NMDAR mediated nNOS activation is prevented by LOPC at 15 days. ( A) Calcium accumulation in the thalamus is not affected by LOPC at all pixel intensities as shown by cumulative tresholding analysis (scalebar = 400 μm). ( B ) The thalamic expression NR1, N2B and nNOS do not change in time or with treatment as assessed by WB. At 15 days, WB analysis show that the expression of synaptic marker PSD-95 is decreased by 50% in LOPC while Synapsin 1 expression is unchanged. ( C ) Co-immunoprecipitation of N2B and PSD-95 shows decreased interaction in LOPC samples between NMDAR and PSD-95 at 15 days (-Ab: beads not coated incubated in lysate; -Lysate: beads coated incubated in PBS). Images cropped from Supp. Fig. 3 . Results are shown as the mean ± SD. *p

    Techniques Used: Activation Assay, Expressing, Western Blot, Marker, Immunoprecipitation, Incubation

    24) Product Images from "Telomere length and telomerase activity in T cells are biomarkers of high‐performing centenarians, et al. Telomere length and telomerase activity in T cells are biomarkers of high‐performing centenarians"

    Article Title: Telomere length and telomerase activity in T cells are biomarkers of high‐performing centenarians, et al. Telomere length and telomerase activity in T cells are biomarkers of high‐performing centenarians

    Journal: Aging Cell

    doi: 10.1111/acel.12859

    Telomerase activity and proliferation capacity in stimulated peripheral blood mononuclear cell (PBMC). (a) Telomerase activity measured by ddTRAP on stimulated PBMC at Days 0, 1, 3, 5, 7, and 10 after stimulation with anti‐CD3/anti‐CD28 Dynabeads. * p
    Figure Legend Snippet: Telomerase activity and proliferation capacity in stimulated peripheral blood mononuclear cell (PBMC). (a) Telomerase activity measured by ddTRAP on stimulated PBMC at Days 0, 1, 3, 5, 7, and 10 after stimulation with anti‐CD3/anti‐CD28 Dynabeads. * p

    Techniques Used: Activity Assay

    25) Product Images from "Ex vivo AKT-inhibition facilitates generation of polyfunctional stem cell memory-like CD8+ T cells for adoptive immunotherapy"

    Article Title: Ex vivo AKT-inhibition facilitates generation of polyfunctional stem cell memory-like CD8+ T cells for adoptive immunotherapy

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2018.1488565

    AKT-inhibition preserves early memory CD8 + T cells with minimal effect on viability and proliferation. CD8 + T N cells were stimulated with CD3/CD28 Dynabeads® in presence of DMSO (Ctrl) or AktiVIII (12 µM), MK (10–5–2.5–1.25 µM), TCN (80–40–20–10 µM), AZD (40–20–10–5 µM), GDC (40–20–10–5 µM), GSK1 (20-10–5-2.5 µM) or GSK2 (40-20-10–5 µM). Viability, proliferation and phenotype were analyzed in 2 independent donors. (A) Percentage viable cells and (B) median fluorescence intensity (MFI) of cell proliferation dye. (C) Representative plots (donor 2) of CD62L expression and cell proliferation dye dilution of Ctrl, 12µM AktiVIII, 5 µM MK, 40 µM TCN, 20 µM AZD, 20 µM GDC, 10 µM GSK1 and 20 µM GSK2-treated T cells, gated on viable CD8 + T cells. Numbers indicate MFI of CD62L. (D) CD62L, CCR7 and CXCR4 expression of CD8 + T cells (Mean + SD, n = 2). Statistical analysis was performed using One-way ANOVA followed by Bonferroni’s Multiple Comparison Test of AKT-inhibited versus Ctrl T cells. ND = not determined, ǂ p
    Figure Legend Snippet: AKT-inhibition preserves early memory CD8 + T cells with minimal effect on viability and proliferation. CD8 + T N cells were stimulated with CD3/CD28 Dynabeads® in presence of DMSO (Ctrl) or AktiVIII (12 µM), MK (10–5–2.5–1.25 µM), TCN (80–40–20–10 µM), AZD (40–20–10–5 µM), GDC (40–20–10–5 µM), GSK1 (20-10–5-2.5 µM) or GSK2 (40-20-10–5 µM). Viability, proliferation and phenotype were analyzed in 2 independent donors. (A) Percentage viable cells and (B) median fluorescence intensity (MFI) of cell proliferation dye. (C) Representative plots (donor 2) of CD62L expression and cell proliferation dye dilution of Ctrl, 12µM AktiVIII, 5 µM MK, 40 µM TCN, 20 µM AZD, 20 µM GDC, 10 µM GSK1 and 20 µM GSK2-treated T cells, gated on viable CD8 + T cells. Numbers indicate MFI of CD62L. (D) CD62L, CCR7 and CXCR4 expression of CD8 + T cells (Mean + SD, n = 2). Statistical analysis was performed using One-way ANOVA followed by Bonferroni’s Multiple Comparison Test of AKT-inhibited versus Ctrl T cells. ND = not determined, ǂ p

    Techniques Used: Inhibition, Fluorescence, Expressing

    26) Product Images from "Distinctive Surface Glycosylation Patterns Associated With Mouse and Human CD4+ Regulatory T Cells and Their Suppressive Function"

    Article Title: Distinctive Surface Glycosylation Patterns Associated With Mouse and Human CD4+ Regulatory T Cells and Their Suppressive Function

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00987

    Flow cytometric analysis of the suppression of CD4 + responder T-cell proliferation and immune cell interactions established by PNGase F-treated Treg in the absence of antigen presenting cells. (A–E) Fluorescence-activated cell sorting (FACS)-purified mouse CD4 + Tconv were cultured alone (0:1) or cocultured in the presence of no enzyme or PNGase F-treated Treg at Treg: responder T-cell ratios of 0:1 and 1:2 for 4 days (A,B) or 8 h (C–E) with anti-CD3/CD28 Dynabeads ® stimulation. (A) Proliferation of CellTrace™ Violet-labeled CD4 + Tconv incubated under different conditions and analyzed by flow cytometry. (B) Graph of the suppressive potency of no enzyme and PNGase F-treated Treg on CD4 + T-cell responders. Suppressive function was quantified based on responder T-cell division index (DI) and presented as the calculated percent suppression [%Suppression (DI)]. For evaluation of immune cell interactions in 8 h cocultures, FACS-purified cells were fluorescently labeled as follows: Treg with CellTrace™ CFSE and CD4 + Tconv with CellTrace™ Far Red DDAO-SE. (C–E) Flow cytometric quantification of the interactions present in the cocultures at 8 h presented as (C) a graph of the overall frequency of multi-cell aggregates and (D,E) pie charts showing the calculated frequencies of the different types of multi-cell aggregates present in the cocultures with (D) no enzyme and (E) PNGase F-treated Treg. Data represent mean ± SD ( n = 3 technical replicates). Statistical analysis was performed by permutation test with an unpaired design (* p value ≤ 0.1).
    Figure Legend Snippet: Flow cytometric analysis of the suppression of CD4 + responder T-cell proliferation and immune cell interactions established by PNGase F-treated Treg in the absence of antigen presenting cells. (A–E) Fluorescence-activated cell sorting (FACS)-purified mouse CD4 + Tconv were cultured alone (0:1) or cocultured in the presence of no enzyme or PNGase F-treated Treg at Treg: responder T-cell ratios of 0:1 and 1:2 for 4 days (A,B) or 8 h (C–E) with anti-CD3/CD28 Dynabeads ® stimulation. (A) Proliferation of CellTrace™ Violet-labeled CD4 + Tconv incubated under different conditions and analyzed by flow cytometry. (B) Graph of the suppressive potency of no enzyme and PNGase F-treated Treg on CD4 + T-cell responders. Suppressive function was quantified based on responder T-cell division index (DI) and presented as the calculated percent suppression [%Suppression (DI)]. For evaluation of immune cell interactions in 8 h cocultures, FACS-purified cells were fluorescently labeled as follows: Treg with CellTrace™ CFSE and CD4 + Tconv with CellTrace™ Far Red DDAO-SE. (C–E) Flow cytometric quantification of the interactions present in the cocultures at 8 h presented as (C) a graph of the overall frequency of multi-cell aggregates and (D,E) pie charts showing the calculated frequencies of the different types of multi-cell aggregates present in the cocultures with (D) no enzyme and (E) PNGase F-treated Treg. Data represent mean ± SD ( n = 3 technical replicates). Statistical analysis was performed by permutation test with an unpaired design (* p value ≤ 0.1).

    Techniques Used: Flow Cytometry, Fluorescence, FACS, Purification, Cell Culture, Labeling, Incubation, Cytometry

    27) Product Images from "A Subset of CXCR5+CD8+ T Cells in the Germinal Centers From Human Tonsils and Lymph Nodes Help B Cells Produce Immunoglobulins"

    Article Title: A Subset of CXCR5+CD8+ T Cells in the Germinal Centers From Human Tonsils and Lymph Nodes Help B Cells Produce Immunoglobulins

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02287

    CXCR5 + CD8 + T Cells provide help to B cells for the production of immunoglobulins. Physical contact between CD8 + T Cells (red) and B cells (green) in tonsil sections, and scale bars, 50μm. (A , n = 5). Sorted tonsil B cells and sorted CD8 + T Cells at the ratio of 1:1, 1:5, and 1:10 were co-cultured with or without α-CD3/CD28 dynabeads for 10 days (B,C) . Sorted B cells were co-cultured with fresh CD8 + or fixed CD8 + T Cells, fresh CD4 + or fixed CD4 + T Cells at the ratio of 5:1 in the presence of α-CD3/CD28 dynabeads for 10 days (D) . Sorted B cells and CD8 + , CXCR5 + CD8 + , CXCR5 − CD8 + T Cells at the ratio of 5:1 were co-cultured with or without anti-IL-21 and anti-CD40L in the presence of α-CD3/CD28 dynabeads for 10 days (E) . The supernatants from the different co-cultures were analyzed by ELISA for the production of IgG, IgM, and IgA. Data are expressed as the mean ± SD, and compared with Mann–Whitney test. * P
    Figure Legend Snippet: CXCR5 + CD8 + T Cells provide help to B cells for the production of immunoglobulins. Physical contact between CD8 + T Cells (red) and B cells (green) in tonsil sections, and scale bars, 50μm. (A , n = 5). Sorted tonsil B cells and sorted CD8 + T Cells at the ratio of 1:1, 1:5, and 1:10 were co-cultured with or without α-CD3/CD28 dynabeads for 10 days (B,C) . Sorted B cells were co-cultured with fresh CD8 + or fixed CD8 + T Cells, fresh CD4 + or fixed CD4 + T Cells at the ratio of 5:1 in the presence of α-CD3/CD28 dynabeads for 10 days (D) . Sorted B cells and CD8 + , CXCR5 + CD8 + , CXCR5 − CD8 + T Cells at the ratio of 5:1 were co-cultured with or without anti-IL-21 and anti-CD40L in the presence of α-CD3/CD28 dynabeads for 10 days (E) . The supernatants from the different co-cultures were analyzed by ELISA for the production of IgG, IgM, and IgA. Data are expressed as the mean ± SD, and compared with Mann–Whitney test. * P

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Expression of co-stimulated molecules on CXCR5 + CD8 + memory T cells from tonsils, lymph nodes and PBMCs. Upon stimulation with CD3/CD28 dynabeads for 8 h, the expression of CD40L and ICOS on CXCR5 + and CXCR5 − CD8 + memory T cells from tonsils, lymph nodes and PBMCs was detected by flow cytometry (A,B) . The data are representative of thirteen or fifteen independent experiments, and were analyzed by two-tailed unpaired t -test (C) . Error bar denote s.e.m. * P
    Figure Legend Snippet: Expression of co-stimulated molecules on CXCR5 + CD8 + memory T cells from tonsils, lymph nodes and PBMCs. Upon stimulation with CD3/CD28 dynabeads for 8 h, the expression of CD40L and ICOS on CXCR5 + and CXCR5 − CD8 + memory T cells from tonsils, lymph nodes and PBMCs was detected by flow cytometry (A,B) . The data are representative of thirteen or fifteen independent experiments, and were analyzed by two-tailed unpaired t -test (C) . Error bar denote s.e.m. * P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Two Tailed Test

    28) Product Images from "PSG9 Stimulates Increase in FoxP3+ Regulatory T-Cells through the TGF-β1 Pathway"

    Article Title: PSG9 Stimulates Increase in FoxP3+ Regulatory T-Cells through the TGF-β1 Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158050

    Administration of PSG9 to naive mouse CD4 + T-cells inhibits IL-2 expression. (A) Cells collected from normal C57BL/6 mouse spleens were treated with equimolar concentration of PSG9 or FLAG control protein and activated with mouse CD3/CD28 T-cell activator Dynabeads. IL-2 levels were determined by ELISA 72 h after activation. P value was calculated using the Student’s t -test, P
    Figure Legend Snippet: Administration of PSG9 to naive mouse CD4 + T-cells inhibits IL-2 expression. (A) Cells collected from normal C57BL/6 mouse spleens were treated with equimolar concentration of PSG9 or FLAG control protein and activated with mouse CD3/CD28 T-cell activator Dynabeads. IL-2 levels were determined by ELISA 72 h after activation. P value was calculated using the Student’s t -test, P

    Techniques Used: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activation Assay

    PSG9 induces the expression of a CD4 + LAP + FoxP3 - regulatory T-cell subset. (A) Human CD4 + naive T-cells were isolated from peripheral blood and plated with human T-cell CD3/CD28 T-cell activator Dynabeads in the presence of IL-2 and PSG9 or FLAG control protein. Expression of CD4 + FoxP3 + LAP + cells was analyzed at 72 h. (B) Graphical representation of the flow cytometry data shown in part C, P
    Figure Legend Snippet: PSG9 induces the expression of a CD4 + LAP + FoxP3 - regulatory T-cell subset. (A) Human CD4 + naive T-cells were isolated from peripheral blood and plated with human T-cell CD3/CD28 T-cell activator Dynabeads in the presence of IL-2 and PSG9 or FLAG control protein. Expression of CD4 + FoxP3 + LAP + cells was analyzed at 72 h. (B) Graphical representation of the flow cytometry data shown in part C, P

    Techniques Used: Expressing, Isolation, Flow Cytometry, Cytometry

    29) Product Images from "Immune Response-Dependent Assembly of IMP Dehydrogenase Filaments"

    Article Title: Immune Response-Dependent Assembly of IMP Dehydrogenase Filaments

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02789

    IMPDH forms filaments during ex vivo primary human T cell activation. (A) Representative images of T cells left untreated or stimulated by mitogens PHA, ConA, or anti-CD3/CD28 for 72 h, then fixed and stained for IMPDH (green) and T cell marker CD3 (red). White arrows: examples of IMPDH filaments. Yellow arrows: example of a ring-shaped filament. Images were captured using identical microscope settings for all treatment groups. (B) Quantification of the percentage of T cells that form filaments when untreated or treated with mitogens PHA, ConA, or anti-CD3/CD28. Cells were cultured in RPMI 1640 under four different conditions: 2 mM glutamine (Gln), 2 mM Gln + 1 h fresh medium, 16 mM Gln, or 16 mM Gln + 1 h fresh medium (represented by differently shaded bars). Different culture conditions were grouped together according to mitogenic treatment and compared to untreated cells (e.g., all PHA-treated conditions grouped vs. all untreated conditions grouped) and statistical significance displayed above each group. No significant differences were observed within these treatment groups due to culture conditions (e.g., PHA 2 mM Gln vs. PHA 16 mM Gln, no difference). Statistical test used: two-way ANOVA followed by Tukey's multiple comparisons test; **** p
    Figure Legend Snippet: IMPDH forms filaments during ex vivo primary human T cell activation. (A) Representative images of T cells left untreated or stimulated by mitogens PHA, ConA, or anti-CD3/CD28 for 72 h, then fixed and stained for IMPDH (green) and T cell marker CD3 (red). White arrows: examples of IMPDH filaments. Yellow arrows: example of a ring-shaped filament. Images were captured using identical microscope settings for all treatment groups. (B) Quantification of the percentage of T cells that form filaments when untreated or treated with mitogens PHA, ConA, or anti-CD3/CD28. Cells were cultured in RPMI 1640 under four different conditions: 2 mM glutamine (Gln), 2 mM Gln + 1 h fresh medium, 16 mM Gln, or 16 mM Gln + 1 h fresh medium (represented by differently shaded bars). Different culture conditions were grouped together according to mitogenic treatment and compared to untreated cells (e.g., all PHA-treated conditions grouped vs. all untreated conditions grouped) and statistical significance displayed above each group. No significant differences were observed within these treatment groups due to culture conditions (e.g., PHA 2 mM Gln vs. PHA 16 mM Gln, no difference). Statistical test used: two-way ANOVA followed by Tukey's multiple comparisons test; **** p

    Techniques Used: Ex Vivo, Activation Assay, Staining, Marker, Microscopy, Cell Culture

    30) Product Images from "Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments"

    Article Title: Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.03.084

    Generation of a Chemically Defined, Customizable Media that Can Expand Human T Cell Subsets in the Absence of Serum (A) The first phase of generating a serum-free medium that can expand all human T cell subsets consisted of creating 10 prototype media by mixing different ratios of 3 base media that contain different concentrations of amino acids, vitamins, trace elements, antioxidants, metal ions, polyamines, and lipids. These prototype media were tested for their ability to expand activated primary human T cells using anti-CD3/CD28-coated beads and reiterated with a design of experiments (DOE) statistical quadratic model through Design-Expert 9.0.1 software with a desired response to maximally expand human T cells without serum supplementation. Phase 2 consisted of eliminating xenogeneic components, examining metabolites consumed in serum-supplemented X-VIVO-15 medium and prototype media from phase 1, and modifying media so that concentrations of metabolites are maintained upon feeding of activated T cells. The final phase focused on optimizing carbon sources, lipid concentrations, lentiviral transduction efficiency, and cytokine production post-activation on activated human T cells. (B) Total CD4 T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and activated by anti-CD3/CD28-coated beads in 1B2H medium containing optimal glucose, no glucose, or optimal glucose without glutamine. T cell proliferation was measured by CFSE dilution by flow cytometry. Data are representative of 3 independent experiments. (C–E) Primary human CD4 T cells were sort-purified into T N (CD25 − CD45RA + CCR7 + CD27 + ) (C), T CM (CD25 − CD45RO + CCR7 + CD27 + ) (D), and T EM cells (CD25 − CD45RO + CCR7 − CD27 − ) (E) and stimulated with anti-CD3/CD28-coated beads in 1B2H medium with or without 5% human serum (see Figure S2 for gating strategy). Cell expansion was monitored by Coulter counter on the indicated days. Data are representative of 2–3 donors and independent experiments. *p
    Figure Legend Snippet: Generation of a Chemically Defined, Customizable Media that Can Expand Human T Cell Subsets in the Absence of Serum (A) The first phase of generating a serum-free medium that can expand all human T cell subsets consisted of creating 10 prototype media by mixing different ratios of 3 base media that contain different concentrations of amino acids, vitamins, trace elements, antioxidants, metal ions, polyamines, and lipids. These prototype media were tested for their ability to expand activated primary human T cells using anti-CD3/CD28-coated beads and reiterated with a design of experiments (DOE) statistical quadratic model through Design-Expert 9.0.1 software with a desired response to maximally expand human T cells without serum supplementation. Phase 2 consisted of eliminating xenogeneic components, examining metabolites consumed in serum-supplemented X-VIVO-15 medium and prototype media from phase 1, and modifying media so that concentrations of metabolites are maintained upon feeding of activated T cells. The final phase focused on optimizing carbon sources, lipid concentrations, lentiviral transduction efficiency, and cytokine production post-activation on activated human T cells. (B) Total CD4 T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and activated by anti-CD3/CD28-coated beads in 1B2H medium containing optimal glucose, no glucose, or optimal glucose without glutamine. T cell proliferation was measured by CFSE dilution by flow cytometry. Data are representative of 3 independent experiments. (C–E) Primary human CD4 T cells were sort-purified into T N (CD25 − CD45RA + CCR7 + CD27 + ) (C), T CM (CD25 − CD45RO + CCR7 + CD27 + ) (D), and T EM cells (CD25 − CD45RO + CCR7 − CD27 − ) (E) and stimulated with anti-CD3/CD28-coated beads in 1B2H medium with or without 5% human serum (see Figure S2 for gating strategy). Cell expansion was monitored by Coulter counter on the indicated days. Data are representative of 2–3 donors and independent experiments. *p

    Techniques Used: Software, Transduction, Activation Assay, Labeling, Flow Cytometry, Cytometry, Purification

    31) Product Images from "Soluble PD-1 ligands regulate T-cell function in Waldenstrom macroglobulinemia"

    Article Title: Soluble PD-1 ligands regulate T-cell function in Waldenstrom macroglobulinemia

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2018021113

    T-cell incubation with the media secreted by WM cells overexpressing PD-L1 and PD-L2 reduces T-cell proliferation and cell cycle proliferation. MWCL-1 cells were transfected with control EV, PD-L1, or PD-L2 constructs. (A) Histograms represent the flow cytometry analysis of the cells overexpressing either PD-L1 (left) or PD-L2 (right). Western blot analysis shows the overexpression of PD-L1 and PD-L2 on the cell surface and in the condition media of the cells, respectively. (B) T cells were isolated from PBMCs and incubated with cell-free media from MWCL-1 lines transfected with overexpressing PD-L1 or PD-L2 constructs. Cells were left either nonstimulated or stimulated with suboptimal (0.5 μg/mL) or optimal (5 μg/mL) dose of CD3 (0.5 μg/mL) and CD28. EV-transfected cells were used as control. Proliferation assay was performed using [ 3 H]TdR following 72 hours of incubation. (C) Western blot analysis was performed on the T-cell lysates after 72 hours of incubation with the media from MWCL-1 lines. (D) Respiratory capacity of T cells in response to treatment with soluble PD-L1 and PD-L2. T cells were stimulated with CD3/CD28 dynabeads for 3 days, and then incubated with cell free–conditioned medias containing soluble PD-L1 and PD-L2 for 1 day. Respiratory capacity of T cells was measured using seahorse XFp analyzer. The diagram is a representative of 3 independent experiments.
    Figure Legend Snippet: T-cell incubation with the media secreted by WM cells overexpressing PD-L1 and PD-L2 reduces T-cell proliferation and cell cycle proliferation. MWCL-1 cells were transfected with control EV, PD-L1, or PD-L2 constructs. (A) Histograms represent the flow cytometry analysis of the cells overexpressing either PD-L1 (left) or PD-L2 (right). Western blot analysis shows the overexpression of PD-L1 and PD-L2 on the cell surface and in the condition media of the cells, respectively. (B) T cells were isolated from PBMCs and incubated with cell-free media from MWCL-1 lines transfected with overexpressing PD-L1 or PD-L2 constructs. Cells were left either nonstimulated or stimulated with suboptimal (0.5 μg/mL) or optimal (5 μg/mL) dose of CD3 (0.5 μg/mL) and CD28. EV-transfected cells were used as control. Proliferation assay was performed using [ 3 H]TdR following 72 hours of incubation. (C) Western blot analysis was performed on the T-cell lysates after 72 hours of incubation with the media from MWCL-1 lines. (D) Respiratory capacity of T cells in response to treatment with soluble PD-L1 and PD-L2. T cells were stimulated with CD3/CD28 dynabeads for 3 days, and then incubated with cell free–conditioned medias containing soluble PD-L1 and PD-L2 for 1 day. Respiratory capacity of T cells was measured using seahorse XFp analyzer. The diagram is a representative of 3 independent experiments.

    Techniques Used: Incubation, Transfection, Construct, Flow Cytometry, Cytometry, Western Blot, Over Expression, Isolation, Proliferation Assay

    32) Product Images from "Discovery and characterization of potent IL-21 neutralizing antibodies via a novel alternating antigen immunization and humanization strategy"

    Article Title: Discovery and characterization of potent IL-21 neutralizing antibodies via a novel alternating antigen immunization and humanization strategy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211236

    19E3 inhibition of PC differentiation mediated by recombinant and endogenous IL-21. (A, B) B cells were isolated from human peripheral blood and stimulated with or without recombinant IL-21 in combination with anti-CD40 and anti-IgM. 19E3 was included at the indicated concentrations. (A) PCs were quantified on day 7 by flow cytometry as IgD – CD38 hi cells (percent of PCs in well indicated). All samples were run in duplicate. (B) Supernatants were collected on day 7 and IgG levels were quantified by ELISA. The experiment was performed with 5 unique donors in 3 separate experiments. Results from a representative donor are shown. (C, D and E) Human B cells were co-cultured with anti-CD3/anti-CD28–activated CD4 + T cells in the presence of graded doses of 19E3. After 7 days of co-culture, PCs, defined as CD19 + IgD - CD38 hi cells, were quantified by flow cytometry. Percent (C, D) and number (E) of PCs for each condition are shown. The experiment was performed with 3 unique donors in 2 separate experiments. Results from 1 representative donor are shown. (F) Human T cells were stimulated with recombinant human IL-21 in combination with anti-CD3. 19E3 was added at the indicated concentrations and T-cell expansion was quantified on day 4. All conditions were run in duplicate. The experiment was performed with 2 unique donors. Results from a representative donor are shown.
    Figure Legend Snippet: 19E3 inhibition of PC differentiation mediated by recombinant and endogenous IL-21. (A, B) B cells were isolated from human peripheral blood and stimulated with or without recombinant IL-21 in combination with anti-CD40 and anti-IgM. 19E3 was included at the indicated concentrations. (A) PCs were quantified on day 7 by flow cytometry as IgD – CD38 hi cells (percent of PCs in well indicated). All samples were run in duplicate. (B) Supernatants were collected on day 7 and IgG levels were quantified by ELISA. The experiment was performed with 5 unique donors in 3 separate experiments. Results from a representative donor are shown. (C, D and E) Human B cells were co-cultured with anti-CD3/anti-CD28–activated CD4 + T cells in the presence of graded doses of 19E3. After 7 days of co-culture, PCs, defined as CD19 + IgD - CD38 hi cells, were quantified by flow cytometry. Percent (C, D) and number (E) of PCs for each condition are shown. The experiment was performed with 3 unique donors in 2 separate experiments. Results from 1 representative donor are shown. (F) Human T cells were stimulated with recombinant human IL-21 in combination with anti-CD3. 19E3 was added at the indicated concentrations and T-cell expansion was quantified on day 4. All conditions were run in duplicate. The experiment was performed with 2 unique donors. Results from a representative donor are shown.

    Techniques Used: Inhibition, Recombinant, Isolation, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay

    33) Product Images from "Tumor microenvironment dictates regulatory T cell phenotype: Upregulated immune checkpoints reinforce suppressive function"

    Article Title: Tumor microenvironment dictates regulatory T cell phenotype: Upregulated immune checkpoints reinforce suppressive function

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1186/s40425-019-0785-8

    Enhanced suppressive function of PD-1-expressing tumor-infiltrating T reg . a Enhanced suppression of CD8 + T cells by PD-1-expressing tumor-infiltrating T reg . At 3 weeks after intravenous injection of TC-1 cells, T reg were isolated from the spleen (SP) and lung of mice with TC-1 cell-induced tumors. SP T reg and tumor-infiltrating T reg expressed low and high levels of PD-1, respectively. CellTrace Violet (CTV)-labeled CD8 + T cells were stimulated in vitro with CD3/CD28 Dynabeads for 72 h in the absence or presence of each T reg population. CTV dilution in proliferating CD8 + T cells is indicated in each histogram. Histograms represent the percentages of proliferating (upper) and IFN-γ-producing (lower) cells. b Contact-dependent T reg -mediated suppression of CD8 + T proliferation. CTV-labeled CD8 + T cells were stimulated in vitro with CD3/CD28 Dynabeads and cocultured with tumor-infiltrating T reg for 72 h in the absence or presence of a transwell membrane. c Homeostatic proliferation of donor Ly5.1 + CD8 + T cells in the spleen isolated from Rag2 −/− mice at 7 d after adoptive cell transfer. Representative plot (left) and absolute number (right) of donor Ly5.1 + CD8 + T cells in the spleen. d PD-1-mediated suppressive activity of tumor-infiltrating T reg isolated from the lungs of tumor-bearing mice 2 weeks after intravenous injection of TC-1 cells. At this time point, T reg expressed intermediate levels of PD-1. CTV-labeled CD8 + T cells were stimulated as shown in ( a ). Before co-culture of CD8 + T cells with tumor-infiltrating T reg , the latter were pre-incubated with an anti-PD-1 antibody or its isotype as control. CTV dilution in proliferating CD8 + T cells is shown in the histograms, which represent the percentages of proliferating (upper) and IFN-γ-producing (lower) cells. ( e ) Representative immunofluorescence images of mouse lung tumor samples. Antibodies against Foxp3, CD8, and PD-1 were used to label and examine the interaction between T reg and CD8 + T cells expressing PD-1. Data are representative of two independent experiments. * P
    Figure Legend Snippet: Enhanced suppressive function of PD-1-expressing tumor-infiltrating T reg . a Enhanced suppression of CD8 + T cells by PD-1-expressing tumor-infiltrating T reg . At 3 weeks after intravenous injection of TC-1 cells, T reg were isolated from the spleen (SP) and lung of mice with TC-1 cell-induced tumors. SP T reg and tumor-infiltrating T reg expressed low and high levels of PD-1, respectively. CellTrace Violet (CTV)-labeled CD8 + T cells were stimulated in vitro with CD3/CD28 Dynabeads for 72 h in the absence or presence of each T reg population. CTV dilution in proliferating CD8 + T cells is indicated in each histogram. Histograms represent the percentages of proliferating (upper) and IFN-γ-producing (lower) cells. b Contact-dependent T reg -mediated suppression of CD8 + T proliferation. CTV-labeled CD8 + T cells were stimulated in vitro with CD3/CD28 Dynabeads and cocultured with tumor-infiltrating T reg for 72 h in the absence or presence of a transwell membrane. c Homeostatic proliferation of donor Ly5.1 + CD8 + T cells in the spleen isolated from Rag2 −/− mice at 7 d after adoptive cell transfer. Representative plot (left) and absolute number (right) of donor Ly5.1 + CD8 + T cells in the spleen. d PD-1-mediated suppressive activity of tumor-infiltrating T reg isolated from the lungs of tumor-bearing mice 2 weeks after intravenous injection of TC-1 cells. At this time point, T reg expressed intermediate levels of PD-1. CTV-labeled CD8 + T cells were stimulated as shown in ( a ). Before co-culture of CD8 + T cells with tumor-infiltrating T reg , the latter were pre-incubated with an anti-PD-1 antibody or its isotype as control. CTV dilution in proliferating CD8 + T cells is shown in the histograms, which represent the percentages of proliferating (upper) and IFN-γ-producing (lower) cells. ( e ) Representative immunofluorescence images of mouse lung tumor samples. Antibodies against Foxp3, CD8, and PD-1 were used to label and examine the interaction between T reg and CD8 + T cells expressing PD-1. Data are representative of two independent experiments. * P

    Techniques Used: Expressing, Injection, Isolation, Mouse Assay, Labeling, In Vitro, Activity Assay, Co-Culture Assay, Incubation, Immunofluorescence

    PD-1-expressing tumor-infiltrating T reg and their activated phenotype in patients with non-small cell lung cancer (NSCLC). a CD25 and Foxp3 expression in CD4 + T cells (upper) and proportion of Foxp3 + cells among total CD4 + T cells (lower) in peripheral blood lymphocytes (PBLs), peritumoral infiltrating lymphocytes (pTILs), and tumor-infiltrating lymphocytes (TILs) derived from patients with NSCLC. b Representative plots of PD-1, TIM-3, TIGIT, CTLA-4, and Foxp3 expression in CD4 + T cells (left) and percentage of CD4 + T cells co-expressing PD-1, TIM-3, TIGIT, CTLA-4, and Foxp3 (right). c PD-1, TIM-3, TIGIT, and CTLA-4 expression in Foxp3 + T reg , Foxp3 − T conv and CD8 + T conv of these patients. d Enhanced suppression of CD8 + T cells by PD-1-expressing tumor-infiltrating T reg from NSCLC patients. T reg were isolated from the peripheral blood and tumor tissue from NSCLC patients. Peripheral blood T reg and tumor-infiltrating T reg expressed low and high levels of PD-1, respectively. CellTrace Violet (CTV)-labeled CD8 + T cells were stimulated in vitro with CD3/CD28 Dynabeads for 96 h in the absence or presence of each T reg population. CTV dilution in proliferating CD8 + T cells is indicated in each histogram. Histograms represent the percentages of proliferating cells. Lines in the bar graph represent the mean and mean ± SEM, respectively. ns, not significant; ** P
    Figure Legend Snippet: PD-1-expressing tumor-infiltrating T reg and their activated phenotype in patients with non-small cell lung cancer (NSCLC). a CD25 and Foxp3 expression in CD4 + T cells (upper) and proportion of Foxp3 + cells among total CD4 + T cells (lower) in peripheral blood lymphocytes (PBLs), peritumoral infiltrating lymphocytes (pTILs), and tumor-infiltrating lymphocytes (TILs) derived from patients with NSCLC. b Representative plots of PD-1, TIM-3, TIGIT, CTLA-4, and Foxp3 expression in CD4 + T cells (left) and percentage of CD4 + T cells co-expressing PD-1, TIM-3, TIGIT, CTLA-4, and Foxp3 (right). c PD-1, TIM-3, TIGIT, and CTLA-4 expression in Foxp3 + T reg , Foxp3 − T conv and CD8 + T conv of these patients. d Enhanced suppression of CD8 + T cells by PD-1-expressing tumor-infiltrating T reg from NSCLC patients. T reg were isolated from the peripheral blood and tumor tissue from NSCLC patients. Peripheral blood T reg and tumor-infiltrating T reg expressed low and high levels of PD-1, respectively. CellTrace Violet (CTV)-labeled CD8 + T cells were stimulated in vitro with CD3/CD28 Dynabeads for 96 h in the absence or presence of each T reg population. CTV dilution in proliferating CD8 + T cells is indicated in each histogram. Histograms represent the percentages of proliferating cells. Lines in the bar graph represent the mean and mean ± SEM, respectively. ns, not significant; ** P

    Techniques Used: Expressing, Derivative Assay, Isolation, Labeling, In Vitro

    34) Product Images from "Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p"

    Article Title: Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p

    Journal: Scientific Reports

    doi: 10.1038/srep20407

    Reconstitution of Sey1p-mediated proteoliposomal membrane docking and lipid mixing. ( a ) Coomassie Blue-stained gel showing proteoliposomes bearing Sey1p and the ER-mimicking lipids. ( b ) The Sey1p proteins reconstituted into proteoliposomes retain GTPase activity. GTPase activity of Sey1p proteoliposomes was assayed, as in Fig. 1c . The concentrations of Sey1p were estimated using a protein-to-lipid ratio of 1/500 (mol/mol). ( c ) Schematic representation of the membrane docking assay using streptavidin-coated beads and Sey1p proteoliposomes bearing either biotin-labeled or Rh-labeled PE. ( d ) Sey1p proteins on two opposing membranes mediate GTP-dependent membrane docking. The biotin-labeled Sey1p proteoliposomes, the Rh-labeled Sey1p proteoliposomes, and streptavidin-coated beads were mixed and incubated in RB500 containing 2 mM MgCl 2 and 1 mM GTP (lane 1). The Rh-labeled liposomes that bound to the biotin liposomes were analyzed by measuring the fluorescence of Rh. As a control, protein-free liposomes bearing biotin-PE (lanes 2 and 4) or Rh-PE (lanes 3 and 4), GTPγS (lane 5), and GDP (lane 6) were added to the reactions. ( e ) Schematic representation of the lipid mixing assay used to monitor dequenching of the NBD fluorescence of Sey1p proteoliposomes. ( f ) Sey1p proteins on two opposing membranes induce efficient lipid mixing in the presence of GTP. Lipid mixing was assayed in RB150 containing 1 mM GTP and 2 mM MgCl 2 , with the Rh/NBD-labeled donor Sy1p proteoliposomes and the non-labeled acceptor Sey1p proteoliposomes. ( g ) Sey1p-mediated lipid mixing requires not only GTP binding but also GTP hydrolysis. Lipid mixing was assayed as in ( f ), in the presence of 1 mM GTP, GDP, GTPγS, or ATP. ( h ) Sey1p-mediated lipid mixing strictly depends on the concentration of GTP. Lipid mixing was assayed as in ( f ), in the presence of various concentrations of GTP. ( i–l ) Negative staining electron microscopy analysis of the lipid mixing reactions of reconstituted Sey1p proteoliposomes. The donor and acceptor proteoliposomes bearing Sey1p were mixed, incubated in the presence ( i,k ) or absence ( J,l ) of 1 mM GTP, and negatively stained with 1% uranyl acetate (UA) ( i,j ) or phosphotungstic acid (PTA) ( k,l ). Scale bars: 500 nm.
    Figure Legend Snippet: Reconstitution of Sey1p-mediated proteoliposomal membrane docking and lipid mixing. ( a ) Coomassie Blue-stained gel showing proteoliposomes bearing Sey1p and the ER-mimicking lipids. ( b ) The Sey1p proteins reconstituted into proteoliposomes retain GTPase activity. GTPase activity of Sey1p proteoliposomes was assayed, as in Fig. 1c . The concentrations of Sey1p were estimated using a protein-to-lipid ratio of 1/500 (mol/mol). ( c ) Schematic representation of the membrane docking assay using streptavidin-coated beads and Sey1p proteoliposomes bearing either biotin-labeled or Rh-labeled PE. ( d ) Sey1p proteins on two opposing membranes mediate GTP-dependent membrane docking. The biotin-labeled Sey1p proteoliposomes, the Rh-labeled Sey1p proteoliposomes, and streptavidin-coated beads were mixed and incubated in RB500 containing 2 mM MgCl 2 and 1 mM GTP (lane 1). The Rh-labeled liposomes that bound to the biotin liposomes were analyzed by measuring the fluorescence of Rh. As a control, protein-free liposomes bearing biotin-PE (lanes 2 and 4) or Rh-PE (lanes 3 and 4), GTPγS (lane 5), and GDP (lane 6) were added to the reactions. ( e ) Schematic representation of the lipid mixing assay used to monitor dequenching of the NBD fluorescence of Sey1p proteoliposomes. ( f ) Sey1p proteins on two opposing membranes induce efficient lipid mixing in the presence of GTP. Lipid mixing was assayed in RB150 containing 1 mM GTP and 2 mM MgCl 2 , with the Rh/NBD-labeled donor Sy1p proteoliposomes and the non-labeled acceptor Sey1p proteoliposomes. ( g ) Sey1p-mediated lipid mixing requires not only GTP binding but also GTP hydrolysis. Lipid mixing was assayed as in ( f ), in the presence of 1 mM GTP, GDP, GTPγS, or ATP. ( h ) Sey1p-mediated lipid mixing strictly depends on the concentration of GTP. Lipid mixing was assayed as in ( f ), in the presence of various concentrations of GTP. ( i–l ) Negative staining electron microscopy analysis of the lipid mixing reactions of reconstituted Sey1p proteoliposomes. The donor and acceptor proteoliposomes bearing Sey1p were mixed, incubated in the presence ( i,k ) or absence ( J,l ) of 1 mM GTP, and negatively stained with 1% uranyl acetate (UA) ( i,j ) or phosphotungstic acid (PTA) ( k,l ). Scale bars: 500 nm.

    Techniques Used: Staining, Activity Assay, Docking Assay, Labeling, Incubation, Fluorescence, Binding Assay, Concentration Assay, Negative Staining, Electron Microscopy

    Requirement of a physiological complex lipid composition for Sey1p-mediated membrane docking and lipid mixing. ( a ) Coomassie Blue-stained gel showing the reconstituted Sey1p proteoliposomes used in ( b–f ), which harbored various sets of lipids ( Table 1). (b ) Lipid composition is not critical for Sey1p-mediated membrane docking. Sey1p-dependent proteoliposomal docking was assayed as in Fig. 2d , using the Sey1p proteoliposomes bearing various sets of lipids ( Table 1 ). Data on the Sey1p liposomes with the complete ER-mimicking lipid set (lanes 1 and 2) are the same as the data shown in Fig. 2d . ( c,d ) A complex but physiological lipid composition is vital for Sey1p-mediated proteoliposomal lipid mixing. Lipid mixing was assayed as in Fig. 2f-h , using the Sey1p liposomes bearing various sets of lipids ( Table 1 ), in the presence ( c ) or absence ( d ) of 1 mM GTP. ( e,f ) Specific lipid molecules (PI/PS/PA, ERG, and PE) are required on both opposing membranes for efficient lipid mixing mediated by Sey1p. Lipid mixing was assayed as in ( c ), except using the Sey1p liposomes bearing the complete lipid set for either the donor ( e ) or the acceptor ( f ) liposomes.
    Figure Legend Snippet: Requirement of a physiological complex lipid composition for Sey1p-mediated membrane docking and lipid mixing. ( a ) Coomassie Blue-stained gel showing the reconstituted Sey1p proteoliposomes used in ( b–f ), which harbored various sets of lipids ( Table 1). (b ) Lipid composition is not critical for Sey1p-mediated membrane docking. Sey1p-dependent proteoliposomal docking was assayed as in Fig. 2d , using the Sey1p proteoliposomes bearing various sets of lipids ( Table 1 ). Data on the Sey1p liposomes with the complete ER-mimicking lipid set (lanes 1 and 2) are the same as the data shown in Fig. 2d . ( c,d ) A complex but physiological lipid composition is vital for Sey1p-mediated proteoliposomal lipid mixing. Lipid mixing was assayed as in Fig. 2f-h , using the Sey1p liposomes bearing various sets of lipids ( Table 1 ), in the presence ( c ) or absence ( d ) of 1 mM GTP. ( e,f ) Specific lipid molecules (PI/PS/PA, ERG, and PE) are required on both opposing membranes for efficient lipid mixing mediated by Sey1p. Lipid mixing was assayed as in ( c ), except using the Sey1p liposomes bearing the complete lipid set for either the donor ( e ) or the acceptor ( f ) liposomes.

    Techniques Used: Staining

    35) Product Images from "Wnt/β-catenin signaling regulates VE-cadherin-mediated anastomosis of brain capillaries by counteracting S1pr1 signaling"

    Article Title: Wnt/β-catenin signaling regulates VE-cadherin-mediated anastomosis of brain capillaries by counteracting S1pr1 signaling

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07302-x

    Junctional localization of VE-cadherin and Esama is affected by Wnt signaling inhibition. a Schematic of the zebrafish hindbrain vasculature at 42 hpf. Red box indicates region of analysis. In the following images ( b – d ) half of the boxed region is displayed. b – d Inhibition of Wnt signaling by IWR-1 from 29 hpf ( b ) or by dnTcf expression ( c , d ) strongly reduced expression of VE-cadherin or Esama and ZO-1 at the cell–cell junctions. Immunostaining for VE-cadherin ( b , c ; red) or Esama ( b , d ; red) and ZO-1 ( b , c ; green). Single channel images were displayed in inverted colors for better visualization. b In control embryos (DMSO), VE-cadherin and Esama were detected in cell–cell junctions along the CtAs and in anastomosis rings (arrow). Inhibition of Wnt signaling resulted in reduced staining of VE-cadherin and Esama at the cell junctions, formation of multiple small anastomosis rings (arrows) and ectopic VE-cadherin-positive cell protrusions (asterisks). c , d In control embryos (mCherry iEC ), VE-cadherin and Esama are strongly expressed in cell–cell junctions. Expression of mCherry-dnTcf iEC dramatically reduced VE-cadherin and ZO-1 ( c ) or Esama ( d ) at the cell–cell junctions. e Primary mouse BECs enriched from microvascular fragments from P3 mice exhibited impaired VE-cadherin junction formation following Wnt signaling inhibition by IWR-1 (DMSO: n = 86; IWR: n = 80; N = 4). In contrast, BECs isolated from adult mice had coverage of cell–cell contact sides by VE-cadherin similar to control (DMSO: n = 143; IWR-1: n = 130; N = 4). Immunostaining for VE-cadherin (red), ZO-1 (green), CD31 (white), and DAPI (blue) of cultured primary mouse BECs after treatment with IWR-1 (P3: 10 µM; adult: 20 µM) or DMSO, respectively. Values represent mean ± SD. * p
    Figure Legend Snippet: Junctional localization of VE-cadherin and Esama is affected by Wnt signaling inhibition. a Schematic of the zebrafish hindbrain vasculature at 42 hpf. Red box indicates region of analysis. In the following images ( b – d ) half of the boxed region is displayed. b – d Inhibition of Wnt signaling by IWR-1 from 29 hpf ( b ) or by dnTcf expression ( c , d ) strongly reduced expression of VE-cadherin or Esama and ZO-1 at the cell–cell junctions. Immunostaining for VE-cadherin ( b , c ; red) or Esama ( b , d ; red) and ZO-1 ( b , c ; green). Single channel images were displayed in inverted colors for better visualization. b In control embryos (DMSO), VE-cadherin and Esama were detected in cell–cell junctions along the CtAs and in anastomosis rings (arrow). Inhibition of Wnt signaling resulted in reduced staining of VE-cadherin and Esama at the cell junctions, formation of multiple small anastomosis rings (arrows) and ectopic VE-cadherin-positive cell protrusions (asterisks). c , d In control embryos (mCherry iEC ), VE-cadherin and Esama are strongly expressed in cell–cell junctions. Expression of mCherry-dnTcf iEC dramatically reduced VE-cadherin and ZO-1 ( c ) or Esama ( d ) at the cell–cell junctions. e Primary mouse BECs enriched from microvascular fragments from P3 mice exhibited impaired VE-cadherin junction formation following Wnt signaling inhibition by IWR-1 (DMSO: n = 86; IWR: n = 80; N = 4). In contrast, BECs isolated from adult mice had coverage of cell–cell contact sides by VE-cadherin similar to control (DMSO: n = 143; IWR-1: n = 130; N = 4). Immunostaining for VE-cadherin (red), ZO-1 (green), CD31 (white), and DAPI (blue) of cultured primary mouse BECs after treatment with IWR-1 (P3: 10 µM; adult: 20 µM) or DMSO, respectively. Values represent mean ± SD. * p

    Techniques Used: Inhibition, Expressing, Immunostaining, Staining, Mouse Assay, Isolation, Cell Culture

    36) Product Images from "NextPBM: a platform to study cell-specific transcription factor binding and cooperativity"

    Article Title: NextPBM: a platform to study cell-specific transcription factor binding and cooperativity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz020

    DNA sequence determinants of PU.1-IRF8 cooperative binding. ( A ) Scatterplot of PU.1 binding z -scores obtained from nuclear extract (nextPBM) versus in vitro transcribed/translated (IVT) PU.1 for random background probes ( n = 500), ETS-IRF composite element (EICE) probes ( n = 116), and canonical PU.1 probes ( n = 2499). ( B ) Scatterplot of IRF8 binding z -scores in nuclear extract versus PU.1 binding z-scores in nuclear extract for the same sets of probes as in (A). ( C ) Left – scatterplot of PU.1 binding z-scores in nuclear extract versus IVT PU.1 for probes included in (A) and SNV probes corresponding to the EICE seed probe shown right. Highlighted probes correspond to SNV probes containing variations in either the ETS core half-site (blue), IRF core site (red), or flanking and linker bases (yellow). Right - schematic of EICE seed probe and bases comprising individual sub-elements. ( D ) Sequence logos obtained using a canonical PU.1 seed probe (left column) and a cooperative ETS-IRF composite element (EICE) probe (right column) from nuclear extract (top row) and from IVT PU.1 (bottom row). ( E ) Workflow schematic for identifying cooperative binding sites using nextPBM. 1 – ChIP-seq sites for a given transcription factor of interest (TF1) can be sampled and used to construct probes for a microarray design. The sample will contain sites where TF1 is cooperatively bound with other factors. 2 – TF1 sample probes are combined with a set of random background probes against which binding z -scores are computed to form the basis of a microarray design. 3 – Profiling binding of TF1 in nuclear extract versus IVT allows for the discovery of cooperative binding sites bound higher in nuclear extract (shown above the diagonal). 4 – Cooperative sites identified can be used as seed probes in a subsequent experiment where SNV probes are included in the microarray and profiled. 5 – Binding to SNV probes is used to model and compare seed- and context-specific DNA binding preferences of TF1 to identify composite elements and likely binding partners.
    Figure Legend Snippet: DNA sequence determinants of PU.1-IRF8 cooperative binding. ( A ) Scatterplot of PU.1 binding z -scores obtained from nuclear extract (nextPBM) versus in vitro transcribed/translated (IVT) PU.1 for random background probes ( n = 500), ETS-IRF composite element (EICE) probes ( n = 116), and canonical PU.1 probes ( n = 2499). ( B ) Scatterplot of IRF8 binding z -scores in nuclear extract versus PU.1 binding z-scores in nuclear extract for the same sets of probes as in (A). ( C ) Left – scatterplot of PU.1 binding z-scores in nuclear extract versus IVT PU.1 for probes included in (A) and SNV probes corresponding to the EICE seed probe shown right. Highlighted probes correspond to SNV probes containing variations in either the ETS core half-site (blue), IRF core site (red), or flanking and linker bases (yellow). Right - schematic of EICE seed probe and bases comprising individual sub-elements. ( D ) Sequence logos obtained using a canonical PU.1 seed probe (left column) and a cooperative ETS-IRF composite element (EICE) probe (right column) from nuclear extract (top row) and from IVT PU.1 (bottom row). ( E ) Workflow schematic for identifying cooperative binding sites using nextPBM. 1 – ChIP-seq sites for a given transcription factor of interest (TF1) can be sampled and used to construct probes for a microarray design. The sample will contain sites where TF1 is cooperatively bound with other factors. 2 – TF1 sample probes are combined with a set of random background probes against which binding z -scores are computed to form the basis of a microarray design. 3 – Profiling binding of TF1 in nuclear extract versus IVT allows for the discovery of cooperative binding sites bound higher in nuclear extract (shown above the diagonal). 4 – Cooperative sites identified can be used as seed probes in a subsequent experiment where SNV probes are included in the microarray and profiled. 5 – Binding to SNV probes is used to model and compare seed- and context-specific DNA binding preferences of TF1 to identify composite elements and likely binding partners.

    Techniques Used: Sequencing, Binding Assay, In Vitro, Chromatin Immunoprecipitation, Construct, Microarray

    Nuclear extract protein-binding microarrays (nextPBMs). ( A ) Workflow schematic for the nextPBM protocol. (1) Cultured cells can be stimulated or treated with a drug prior to nuclear extraction. (2) Total soluble protein content is harvested from cell nuclei using an optimized protocol (see Materials and Methods). (3) Nuclear extract can be treated in parallel enzymatically (i.e. by phosphatase treatment) and components of interest can be depleted (i.e. by immune-depletion using a targeted antibody) depending on goals of the experiments. 4) DNA binding affinity of one or more transcription factors of interest are profiled in parallel directly from nuclear extract. ( B ) Density of PU.1 nextPBM z -scores obtained at random background probes ( n = 500) and at genomic PU.1 binding sites ( n = 2615). ( C ) Scatterplot of PU.1 binding z -scores obtained by DNA probes corresponding to random background (black) and genomic PU.1 sites (blue) in different biological replicates. ( D ) Left: Schematic representation of the single nucleotide variant (SNV) probes corresponding to an example PU.1 seed probe. Genomic sequence corresponding to the PU.1 motif is highlighted in sky blue within a larger 20bp sequence. SNVs within a given SNV probe are shown in dark blue. Right: Sequence logos obtained for the same genomic PU.1 seed probe using a PU.1 antibody (top) and an FLI1 antibody (bottom). Δz-scores are computed relative to the median score obtained within a given column.
    Figure Legend Snippet: Nuclear extract protein-binding microarrays (nextPBMs). ( A ) Workflow schematic for the nextPBM protocol. (1) Cultured cells can be stimulated or treated with a drug prior to nuclear extraction. (2) Total soluble protein content is harvested from cell nuclei using an optimized protocol (see Materials and Methods). (3) Nuclear extract can be treated in parallel enzymatically (i.e. by phosphatase treatment) and components of interest can be depleted (i.e. by immune-depletion using a targeted antibody) depending on goals of the experiments. 4) DNA binding affinity of one or more transcription factors of interest are profiled in parallel directly from nuclear extract. ( B ) Density of PU.1 nextPBM z -scores obtained at random background probes ( n = 500) and at genomic PU.1 binding sites ( n = 2615). ( C ) Scatterplot of PU.1 binding z -scores obtained by DNA probes corresponding to random background (black) and genomic PU.1 sites (blue) in different biological replicates. ( D ) Left: Schematic representation of the single nucleotide variant (SNV) probes corresponding to an example PU.1 seed probe. Genomic sequence corresponding to the PU.1 motif is highlighted in sky blue within a larger 20bp sequence. SNVs within a given SNV probe are shown in dark blue. Right: Sequence logos obtained for the same genomic PU.1 seed probe using a PU.1 antibody (top) and an FLI1 antibody (bottom). Δz-scores are computed relative to the median score obtained within a given column.

    Techniques Used: Protein Binding, Cell Culture, Binding Assay, Variant Assay, Sequencing

    37) Product Images from "The Nuclear Envelope Protein, LAP1B, Is a Novel Protein Phosphatase 1 Substrate"

    Article Title: The Nuclear Envelope Protein, LAP1B, Is a Novel Protein Phosphatase 1 Substrate

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076788

    Co-immunoprecipitation of the PP1:LAP1B complex in COS-7 cells, SH-SY5Y cells and rat cortex. A- COS-7 cells were transfected with Myc-LAP1B, Myc-LAP1B-BM2 or Myc-LAP1B-BM3 and immunoprecipitated with PP1γ bound to protein G- DynaBeads. B- Non-transfected COS-7 cells were immunoprecipitated with PP1γ or PP1α antibodies bound to protein G- Dynabeads. C-SH-SY5Y cells were immunoprecipitated with PP1γ or PP1α antibodies bound to protein G- Dynabeads. D- Rat cortex extracts were immunoprecipitated with PP1γ, PP1α or LAP1 antibodies bound to protein G- Dynabeads. The negative controls were performed by incubating cell extracts with beads. IP, immunoprecipitation. IB, immunoblotting.
    Figure Legend Snippet: Co-immunoprecipitation of the PP1:LAP1B complex in COS-7 cells, SH-SY5Y cells and rat cortex. A- COS-7 cells were transfected with Myc-LAP1B, Myc-LAP1B-BM2 or Myc-LAP1B-BM3 and immunoprecipitated with PP1γ bound to protein G- DynaBeads. B- Non-transfected COS-7 cells were immunoprecipitated with PP1γ or PP1α antibodies bound to protein G- Dynabeads. C-SH-SY5Y cells were immunoprecipitated with PP1γ or PP1α antibodies bound to protein G- Dynabeads. D- Rat cortex extracts were immunoprecipitated with PP1γ, PP1α or LAP1 antibodies bound to protein G- Dynabeads. The negative controls were performed by incubating cell extracts with beads. IP, immunoprecipitation. IB, immunoblotting.

    Techniques Used: Immunoprecipitation, Transfection

    38) Product Images from "Cyclin D1 represses peroxisome proliferator-activated receptor alpha and inhibits fatty acid oxidation"

    Article Title: Cyclin D1 represses peroxisome proliferator-activated receptor alpha and inhibits fatty acid oxidation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10274

    Cyclin D1 inhibits PPARα and fatty acid oxidation in liver cancer cells HepG2 cells were cultured in the presence or absence of serum and siRNA as indicted, and harvested after 72 hr. ( A ) Western blot. ( B ) DNA synthesis and cell viability ( C ) PPARα-mediated transcript expression. ( D ) Chromatin Immunoprecipitation (ChIP) using antibodies to PPARa (top) and PolII (bottom). ( E ) Fatty acid oxidation.
    Figure Legend Snippet: Cyclin D1 inhibits PPARα and fatty acid oxidation in liver cancer cells HepG2 cells were cultured in the presence or absence of serum and siRNA as indicted, and harvested after 72 hr. ( A ) Western blot. ( B ) DNA synthesis and cell viability ( C ) PPARα-mediated transcript expression. ( D ) Chromatin Immunoprecipitation (ChIP) using antibodies to PPARa (top) and PolII (bottom). ( E ) Fatty acid oxidation.

    Techniques Used: Cell Culture, Western Blot, DNA Synthesis, Expressing, Chromatin Immunoprecipitation

    39) Product Images from "Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector, Ixodes scapularis"

    Article Title: Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector, Ixodes scapularis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137237

    Characterization of A . phagocytophilum protein-protein interactions. (A) Protein-protein interactions were characterized in silico using STRING 8.3 ( http://string-db.org ). The STRING score value is shown, defined as threshold of significance to include the interaction (maximum value = 1) computed by combining the probabilities from the different evidence channels, correcting for the probability of randomly observing an interaction. (B) Protein-protein interactions were characterized in vitro using A . phagocytophilum HSP70 (red arrow) and GroEL (blue arrow) recombinant proteins and tick Porin as control [ 15 ]. The proteins were mixed in equimolar amounts and immunoprecipitated using anti-GroEL or anti-HSP70 antibodies and Protein G Dynabeads. The purified proteins were eluted using Laemmli sample buffer and loaded onto a 12% SDS-PAGE gel for Western blot analysis using anti-HSP70, anti-GroEL or anti-Porin antibodies. (C) Protein-protein interactions were characterized in vitro using A . phagocytophilum protein extracts, recombinant HSP70 (red arrow) and GroEL (blue arrow) proteins and tick Porin as control [ 15 ]. Protein G Dynabeads were incubated with purified anti-HSP70, anti-GroEL or anti-Porin antibodies and then 130 μg of A . phagocytophilum proteins were added. Unbound proteins were removed and the beads were washed three times with PBS with addition of 0.1% Triton X-100, resuspended in Laemmli sample buffer and loaded onto a 12% SDS-PAGE gel for Western blot analysis using anti-HSP70 or anti-GroEL antibodies. (D) Protein-protein interactions were characterized in vitro using A . phagocytophilum HSP70 (red arrow), GroEL (blue arrow) and MSP4 (green arrows) recombinant proteins and tick Porin as control [ 15 ]. Nickel beads were covered with histidine-tagged MSP4, washed and incubated with GroEL or HSP70, MSP4 or Porin as control. After incubation, beads were washed and proteins eluted in Laemmli sample buffer and loaded onto a 15% SDS-PAGE gel.
    Figure Legend Snippet: Characterization of A . phagocytophilum protein-protein interactions. (A) Protein-protein interactions were characterized in silico using STRING 8.3 ( http://string-db.org ). The STRING score value is shown, defined as threshold of significance to include the interaction (maximum value = 1) computed by combining the probabilities from the different evidence channels, correcting for the probability of randomly observing an interaction. (B) Protein-protein interactions were characterized in vitro using A . phagocytophilum HSP70 (red arrow) and GroEL (blue arrow) recombinant proteins and tick Porin as control [ 15 ]. The proteins were mixed in equimolar amounts and immunoprecipitated using anti-GroEL or anti-HSP70 antibodies and Protein G Dynabeads. The purified proteins were eluted using Laemmli sample buffer and loaded onto a 12% SDS-PAGE gel for Western blot analysis using anti-HSP70, anti-GroEL or anti-Porin antibodies. (C) Protein-protein interactions were characterized in vitro using A . phagocytophilum protein extracts, recombinant HSP70 (red arrow) and GroEL (blue arrow) proteins and tick Porin as control [ 15 ]. Protein G Dynabeads were incubated with purified anti-HSP70, anti-GroEL or anti-Porin antibodies and then 130 μg of A . phagocytophilum proteins were added. Unbound proteins were removed and the beads were washed three times with PBS with addition of 0.1% Triton X-100, resuspended in Laemmli sample buffer and loaded onto a 12% SDS-PAGE gel for Western blot analysis using anti-HSP70 or anti-GroEL antibodies. (D) Protein-protein interactions were characterized in vitro using A . phagocytophilum HSP70 (red arrow), GroEL (blue arrow) and MSP4 (green arrows) recombinant proteins and tick Porin as control [ 15 ]. Nickel beads were covered with histidine-tagged MSP4, washed and incubated with GroEL or HSP70, MSP4 or Porin as control. After incubation, beads were washed and proteins eluted in Laemmli sample buffer and loaded onto a 15% SDS-PAGE gel.

    Techniques Used: In Silico, In Vitro, Recombinant, Immunoprecipitation, Purification, SDS Page, Western Blot, Incubation

    40) Product Images from "Two E3 ligases antagonistically regulate the UV-B response in Arabidopsis"

    Article Title: Two E3 ligases antagonistically regulate the UV-B response in Arabidopsis

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

    doi: 10.1073/pnas.1816268116

    COP1 degrades RUP1 and RUP2 under UV-B light. ( A ) Effect of COP1 on RUP2 stability in Arabidopsis under UV-B light. Immunoblot analysis of RUP2 proteins in 4-d-old Col and cop1-4 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( B ) Effect of COP1 on RUP2 stability in vitro under UV-B light, as analyzed by cell-free degradation assays. Purified His-RUP2 was incubated with total proteins extracted from 4-d-old Col and cop1-4 seedlings grown under +UV-B light for 2 h. The degradation mixture was treated with or without 50 μM MG132. His-RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( C ) Effect of FLAG-COP1 on the ubiquitination of RUP2-HA in HEK293T cells. Total proteins were extracted from HEK293T cells that were transfected with FLAG/FLAG-COP1 and HA/RUP2-HA for co-IP with Dynabeads Protein G and anti-HA antibodies. Proteins were analyzed by immunoblotting with anti-HA and anti-Ubiquitin antibodies. Ubn, ubiquitin chain. The asterisks indicate nonspecific bands. ( D and E ) Effect of COP1 on the ubiquitination of FLAG-RUP1 in vivo. Total proteins were extracted from 4-d-old Col, FLAG-RUP1, FLAG-RUP1/ cop1-4 ( D ), or FLAG-RUP1 YFP-COP1 ( E ) seedlings grown under +UV-B light and treated with 50 μM MG132 for 24 h before co-IP with ANTI-FLAG Magnetic Beads. Proteins were analyzed by immunoblotting with anti-FLAG and anti-Ubiquitin antibodies. The asterisks indicate nonspecific bands.
    Figure Legend Snippet: COP1 degrades RUP1 and RUP2 under UV-B light. ( A ) Effect of COP1 on RUP2 stability in Arabidopsis under UV-B light. Immunoblot analysis of RUP2 proteins in 4-d-old Col and cop1-4 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( B ) Effect of COP1 on RUP2 stability in vitro under UV-B light, as analyzed by cell-free degradation assays. Purified His-RUP2 was incubated with total proteins extracted from 4-d-old Col and cop1-4 seedlings grown under +UV-B light for 2 h. The degradation mixture was treated with or without 50 μM MG132. His-RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( C ) Effect of FLAG-COP1 on the ubiquitination of RUP2-HA in HEK293T cells. Total proteins were extracted from HEK293T cells that were transfected with FLAG/FLAG-COP1 and HA/RUP2-HA for co-IP with Dynabeads Protein G and anti-HA antibodies. Proteins were analyzed by immunoblotting with anti-HA and anti-Ubiquitin antibodies. Ubn, ubiquitin chain. The asterisks indicate nonspecific bands. ( D and E ) Effect of COP1 on the ubiquitination of FLAG-RUP1 in vivo. Total proteins were extracted from 4-d-old Col, FLAG-RUP1, FLAG-RUP1/ cop1-4 ( D ), or FLAG-RUP1 YFP-COP1 ( E ) seedlings grown under +UV-B light and treated with 50 μM MG132 for 24 h before co-IP with ANTI-FLAG Magnetic Beads. Proteins were analyzed by immunoblotting with anti-FLAG and anti-Ubiquitin antibodies. The asterisks indicate nonspecific bands.

    Techniques Used: Negative Control, In Vitro, Purification, Incubation, Transfection, Co-Immunoprecipitation Assay, In Vivo, Magnetic Beads

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    Article Snippet: .. Extracts were cleared by centrifugation at 13,000 g for 10 min, diluted five-fold in immunoprecipitation bufffer and then pre-cleared by incubation with Protein G–Dynabeads (Thermo Scientific) in the absence of antibodies for 2 h at 4°C. .. NMDA-R NR1 subunit-specific antibody (NR1-Ab) (Millipore; 05-432) and non-specific mouse IgG (NS-IgG) were coupled to Protein-G–Dynabeads by incubation for 30 min at 4°C.

    Article Title: Albumin Is Synthesized in Epididymis and Aggregates in a High Molecular Mass Glycoprotein Complex Involved in Sperm-Egg Fertilization
    Article Snippet: .. Immunoprecipitation assays The pAb rabbit anti-albumin IgG (Abcam, Tokyo, Japan; ab34807) or mAb TRA 54 (Abcam, Tokyo, Japan) , was added to Dynabeads-protein G (Invitrogen, Tokyo, Japan), washed three times with washing buffer and cross-linked with dimethyl pimelimidate (DMP) buffer, followed by an additional three washes with 25 mM citrate/52 mM phosphate buffer (pH 5.0), according to the manufacturer’s instructions, prior to storage at 4°C. .. Caput epididymis was homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 M sucrose, 1% dextran, 5 mM MgCl2 ) containing protease inhibitors – 1 mM EDTA and antipain, leupeptin and pepstatin A (3 µg/ml each).

    Centrifugation:

    Article Title: PAI1 blocks NMDA receptor-mediated effects of tissue-type plasminogen activator on cell signaling and physiology
    Article Snippet: .. Extracts were cleared by centrifugation at 13,000 g for 10 min, diluted five-fold in immunoprecipitation bufffer and then pre-cleared by incubation with Protein G–Dynabeads (Thermo Scientific) in the absence of antibodies for 2 h at 4°C. .. NMDA-R NR1 subunit-specific antibody (NR1-Ab) (Millipore; 05-432) and non-specific mouse IgG (NS-IgG) were coupled to Protein-G–Dynabeads by incubation for 30 min at 4°C.

    Article Title: Self-antigen recognition by follicular lymphoma B-cell receptors
    Article Snippet: .. Cells were lysed at 10 × 106 cells/mL with nondenaturing lysis buffer (20mM Tris HCL pH 8, 137mM NaCl, 1% NP-40) containing protease inhibitors (Roche cOmplete Mini, EDTA-free protease inhibitor tablets) at 4°C for 45 minutes followed by centrifugation at 20 000 g for 20 minutes at 4°C; 1 μg of tumor Ig was added to 1 mL of lysate and rotated for 2 hours at room temperature, followed by addition of 25 μL of protein G beads (Dynabeads, Invitrogen), and continued rotation for 15 minutes. .. The beads were washed 5× with PBS and samples were eluted with nonreducing SDS sample buffer, and separated by SDS-PAGE.

    Incubation:

    Article Title: PAI1 blocks NMDA receptor-mediated effects of tissue-type plasminogen activator on cell signaling and physiology
    Article Snippet: .. Extracts were cleared by centrifugation at 13,000 g for 10 min, diluted five-fold in immunoprecipitation bufffer and then pre-cleared by incubation with Protein G–Dynabeads (Thermo Scientific) in the absence of antibodies for 2 h at 4°C. .. NMDA-R NR1 subunit-specific antibody (NR1-Ab) (Millipore; 05-432) and non-specific mouse IgG (NS-IgG) were coupled to Protein-G–Dynabeads by incubation for 30 min at 4°C.

    Protease Inhibitor:

    Article Title: Self-antigen recognition by follicular lymphoma B-cell receptors
    Article Snippet: .. Cells were lysed at 10 × 106 cells/mL with nondenaturing lysis buffer (20mM Tris HCL pH 8, 137mM NaCl, 1% NP-40) containing protease inhibitors (Roche cOmplete Mini, EDTA-free protease inhibitor tablets) at 4°C for 45 minutes followed by centrifugation at 20 000 g for 20 minutes at 4°C; 1 μg of tumor Ig was added to 1 mL of lysate and rotated for 2 hours at room temperature, followed by addition of 25 μL of protein G beads (Dynabeads, Invitrogen), and continued rotation for 15 minutes. .. The beads were washed 5× with PBS and samples were eluted with nonreducing SDS sample buffer, and separated by SDS-PAGE.

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