streptavidin agarose  (Millipore)


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    Name:
    Streptavidin Agarose from Streptomyces avidinii
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    Catalog Number:
    s1638
    Price:
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    Applications:
    Used for the purification of biotin containing proteins or DNA binding proteins
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    Structured Review

    Millipore streptavidin agarose
    Suppression of COX-2 expression and NF-κB binding by lasiodin. ( A ), CNE1 and CNE2 cells were treated with lasiodin at the indicated doses. After 24 hr treatment, the expressions of COX-2, IKK and NF-κB were detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. GAPDH was used as the control for sample loading. ( B ), CNE1 and CNE2 cells were treated with the COX-2-selective inhibitor (celecoxib, 20 µM) for 4 hr, and then treated with lasiodin at the indicated doses. After 48 hr treatment, cell viability was determined by the MTT assay. ( C ), CNE1 and CNE2 cells were treated with lasiodin at 6.3 µM for 24 hr. The binding of the transactivators to the COX-2 promoter was analyzed by the <t>streptavidin-agrose</t> pulldown assay. NPC cells treated with the vehicle control (DMSO) were used as the reference group. ( D ), CNE1 and CNE2 cells were treated with lasiodin at the dose of 6.3 µM for 24 hr. The nuclear extracts were prepared, and NF-κB was detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. ( E ), CNE1 and CNE2 cells were treated with lasiodin at 6.3 µM for 24 hr. The NF-κB nuclear translocations in CNE1 and CNE2 cells were determined by immunofluorescence imaging analysis. NPC cells treated with the vehicle control (DMSO) were used as the reference group. ( F ), The proposed mechanisms were shown. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P

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    Images

    1) Product Images from "Lasiodin Inhibits Proliferation of Human Nasopharyngeal Carcinoma Cells by Simultaneous Modulation of the Apaf-1/Caspase, AKT/MAPK and COX-2/NF-?B Signaling Pathways"

    Article Title: Lasiodin Inhibits Proliferation of Human Nasopharyngeal Carcinoma Cells by Simultaneous Modulation of the Apaf-1/Caspase, AKT/MAPK and COX-2/NF-?B Signaling Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097799

    Suppression of COX-2 expression and NF-κB binding by lasiodin. ( A ), CNE1 and CNE2 cells were treated with lasiodin at the indicated doses. After 24 hr treatment, the expressions of COX-2, IKK and NF-κB were detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. GAPDH was used as the control for sample loading. ( B ), CNE1 and CNE2 cells were treated with the COX-2-selective inhibitor (celecoxib, 20 µM) for 4 hr, and then treated with lasiodin at the indicated doses. After 48 hr treatment, cell viability was determined by the MTT assay. ( C ), CNE1 and CNE2 cells were treated with lasiodin at 6.3 µM for 24 hr. The binding of the transactivators to the COX-2 promoter was analyzed by the streptavidin-agrose pulldown assay. NPC cells treated with the vehicle control (DMSO) were used as the reference group. ( D ), CNE1 and CNE2 cells were treated with lasiodin at the dose of 6.3 µM for 24 hr. The nuclear extracts were prepared, and NF-κB was detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. ( E ), CNE1 and CNE2 cells were treated with lasiodin at 6.3 µM for 24 hr. The NF-κB nuclear translocations in CNE1 and CNE2 cells were determined by immunofluorescence imaging analysis. NPC cells treated with the vehicle control (DMSO) were used as the reference group. ( F ), The proposed mechanisms were shown. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P
    Figure Legend Snippet: Suppression of COX-2 expression and NF-κB binding by lasiodin. ( A ), CNE1 and CNE2 cells were treated with lasiodin at the indicated doses. After 24 hr treatment, the expressions of COX-2, IKK and NF-κB were detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. GAPDH was used as the control for sample loading. ( B ), CNE1 and CNE2 cells were treated with the COX-2-selective inhibitor (celecoxib, 20 µM) for 4 hr, and then treated with lasiodin at the indicated doses. After 48 hr treatment, cell viability was determined by the MTT assay. ( C ), CNE1 and CNE2 cells were treated with lasiodin at 6.3 µM for 24 hr. The binding of the transactivators to the COX-2 promoter was analyzed by the streptavidin-agrose pulldown assay. NPC cells treated with the vehicle control (DMSO) were used as the reference group. ( D ), CNE1 and CNE2 cells were treated with lasiodin at the dose of 6.3 µM for 24 hr. The nuclear extracts were prepared, and NF-κB was detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. ( E ), CNE1 and CNE2 cells were treated with lasiodin at 6.3 µM for 24 hr. The NF-κB nuclear translocations in CNE1 and CNE2 cells were determined by immunofluorescence imaging analysis. NPC cells treated with the vehicle control (DMSO) were used as the reference group. ( F ), The proposed mechanisms were shown. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P

    Techniques Used: Expressing, Binding Assay, Western Blot, MTT Assay, Immunofluorescence, Imaging

    2) Product Images from "Two new mutations in the HIF2A gene associated with erythrocytosis"

    Article Title: Two new mutations in the HIF2A gene associated with erythrocytosis

    Journal: American Journal of Hematology

    doi: 10.1002/ajh.23123

    Binding assays. (A) Recombinant FlagVHL was incubated without (lane 2) or with(lanes 3 to 5) biotinylated Hyp-564 HIF-1α (556–574) peptide prebound to streptavidin-agarose, in the absence(lane 3)or presence of 5 nM wild type (WT) or F540L Hyp-531 HIF-2α (527–542) peptide (lanes 4 and 5, respectively). The resins (lanes 2 to 5) were washed, eluted with 2 × SDS loading buffer, and the eluates subjected to SDS-PAGE and western blotting using anti-Flag antibodies. Quantification was performed using a Chemi Doc-It system. Input represents 1% of the total VHL added to each reaction. The degree of inhibition observed with WT HIF-2α (527–542) peptide was 84 ±1.2% (n = 3), while that observed with the mutant peptide was 34 ±2.8% (n = 3)( P
    Figure Legend Snippet: Binding assays. (A) Recombinant FlagVHL was incubated without (lane 2) or with(lanes 3 to 5) biotinylated Hyp-564 HIF-1α (556–574) peptide prebound to streptavidin-agarose, in the absence(lane 3)or presence of 5 nM wild type (WT) or F540L Hyp-531 HIF-2α (527–542) peptide (lanes 4 and 5, respectively). The resins (lanes 2 to 5) were washed, eluted with 2 × SDS loading buffer, and the eluates subjected to SDS-PAGE and western blotting using anti-Flag antibodies. Quantification was performed using a Chemi Doc-It system. Input represents 1% of the total VHL added to each reaction. The degree of inhibition observed with WT HIF-2α (527–542) peptide was 84 ±1.2% (n = 3), while that observed with the mutant peptide was 34 ±2.8% (n = 3)( P

    Techniques Used: Binding Assay, Recombinant, Incubation, SDS Page, Western Blot, Inhibition, Mutagenesis

    3) Product Images from "Cell-surface Processing of the Metalloprotease Pro-ADAMTS9 Is Influenced by the Chaperone GRP94/gp96 *"

    Article Title: Cell-surface Processing of the Metalloprotease Pro-ADAMTS9 Is Influenced by the Chaperone GRP94/gp96 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.039677

    Cell-surface localization of gp96 and BiP in HEK293F cells. Following cell-surface biotinylation (with (+) or without (−) prior trypsinization), Western blotting of streptavidin-agarose-purified proteins with the indicated antibodies demonstrates
    Figure Legend Snippet: Cell-surface localization of gp96 and BiP in HEK293F cells. Following cell-surface biotinylation (with (+) or without (−) prior trypsinization), Western blotting of streptavidin-agarose-purified proteins with the indicated antibodies demonstrates

    Techniques Used: Western Blot, Purification

    4) Product Images from "RNA affinity tags for purification of RNAs and ribonucleoprotein complexes"

    Article Title: RNA affinity tags for purification of RNAs and ribonucleoprotein complexes

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/S1046-2023(02)00018-X

    Affinity tag insertions into the RNA subunit of ribonuclease P. Both the D8 Sephadex tag and the S1 streptavidin tag have been inserted into the yeast RPR1 ]. Tags were inserted into nonessential loops at the end of stems that were known to protrude into solution. Both insertions gave correct assembly of functional enzyme in vivo, allowing purification of the enzyme from cells containing the modified gene as the only source of RPR1 RNA.
    Figure Legend Snippet: Affinity tag insertions into the RNA subunit of ribonuclease P. Both the D8 Sephadex tag and the S1 streptavidin tag have been inserted into the yeast RPR1 ]. Tags were inserted into nonessential loops at the end of stems that were known to protrude into solution. Both insertions gave correct assembly of functional enzyme in vivo, allowing purification of the enzyme from cells containing the modified gene as the only source of RPR1 RNA.

    Techniques Used: Functional Assay, In Vivo, Purification, Modification

    Minimal binding motifs and consensus structures of Sephadex-and streptavidin-binding RNA affinity tags. The actual sequences of the D8 and S1 tags are shown, along with the consensus structures showing conserved stem and loop regions versus conserved nucleotide identities (× indicates nonconserved nucleotides).
    Figure Legend Snippet: Minimal binding motifs and consensus structures of Sephadex-and streptavidin-binding RNA affinity tags. The actual sequences of the D8 and S1 tags are shown, along with the consensus structures showing conserved stem and loop regions versus conserved nucleotide identities (× indicates nonconserved nucleotides).

    Techniques Used: Binding Assay

    Isolation of RNase P using the S1 streptavidin tag. Soluble extracts from either the wild-type or S1-tagged RPR1 ]. (A) The presence of RPR1 RNA in the input and the eluate fractions was analyzed by Northern blot analysis. Only RNase P containing the S1-tagged RPR1 ].
    Figure Legend Snippet: Isolation of RNase P using the S1 streptavidin tag. Soluble extracts from either the wild-type or S1-tagged RPR1 ]. (A) The presence of RPR1 RNA in the input and the eluate fractions was analyzed by Northern blot analysis. Only RNase P containing the S1-tagged RPR1 ].

    Techniques Used: Isolation, Northern Blot

    5) Product Images from "XIAP upregulates expression of HIF target genes by targeting HIF1α for Lys63-linked polyubiquitination"

    Article Title: XIAP upregulates expression of HIF target genes by targeting HIF1α for Lys63-linked polyubiquitination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx549

    XIAP ubiquitinates HIF1α. ( A ) U2OS cells were transfected with siRNA oligonucleotides and His-tagged ubiquitin as indicated. Lysates were prepared and precipitated using nickel-conjugated beads followed by western blotting using the indicated antibodies. ( B ) HA-tagged plasmid encoding HIF1α was coexpressed in HEK293 cells with His-tagged ubiquitin (Ub), FLAG-XIAP (X) or FLAG-VHL (V) as indicated. Lysates were prepared 48 h post-transfection and ubiquitinated material was recovered by incubation with nickel-conjugated beads and analysed by western blotting. ( C ) Biotinylated-HIF1α was coexpressed in HEK293 cells with FLAG-tagged XIAP. HIF1α was recovered from lysates by incubation with streptavidin-coated beads and analysed using antibodies directed against specific ubiquitin chains. ( D ) U2OS cells were pre-treated with 2μM NSC697923 for 30 min before being exposed to 1% O 2 for 3 h. Cells were lysed in the presence of UBQ-TUBEs. Ubiquitin conjugates were purified using NiNTA agarose beads and analysed by immunoblotting using the indicated antibodies. ( E ) U2OS cells were pre-treated with 2 μM NSC697923 for 30 min before being exposed to 1% O 2 for 3 h. Cells were lysed in the presence of TAB2-TUBEs or TAB2-AA-TUBEs as indicated. Proteins conjugated to Lys 63 -linked ubiquitin chains were purified using Ni-NTA agarose beads and analysed by immunoblot.
    Figure Legend Snippet: XIAP ubiquitinates HIF1α. ( A ) U2OS cells were transfected with siRNA oligonucleotides and His-tagged ubiquitin as indicated. Lysates were prepared and precipitated using nickel-conjugated beads followed by western blotting using the indicated antibodies. ( B ) HA-tagged plasmid encoding HIF1α was coexpressed in HEK293 cells with His-tagged ubiquitin (Ub), FLAG-XIAP (X) or FLAG-VHL (V) as indicated. Lysates were prepared 48 h post-transfection and ubiquitinated material was recovered by incubation with nickel-conjugated beads and analysed by western blotting. ( C ) Biotinylated-HIF1α was coexpressed in HEK293 cells with FLAG-tagged XIAP. HIF1α was recovered from lysates by incubation with streptavidin-coated beads and analysed using antibodies directed against specific ubiquitin chains. ( D ) U2OS cells were pre-treated with 2μM NSC697923 for 30 min before being exposed to 1% O 2 for 3 h. Cells were lysed in the presence of UBQ-TUBEs. Ubiquitin conjugates were purified using NiNTA agarose beads and analysed by immunoblotting using the indicated antibodies. ( E ) U2OS cells were pre-treated with 2 μM NSC697923 for 30 min before being exposed to 1% O 2 for 3 h. Cells were lysed in the presence of TAB2-TUBEs or TAB2-AA-TUBEs as indicated. Proteins conjugated to Lys 63 -linked ubiquitin chains were purified using Ni-NTA agarose beads and analysed by immunoblot.

    Techniques Used: Transfection, Western Blot, Plasmid Preparation, Incubation, Purification

    6) Product Images from "Monoclonal Antibody 16D10 to the C-Terminal Domain of the Feto-Acinar Pancreatic Protein Binds to Membrane of Human Pancreatic Tumoral SOJ-6 Cells and Inhibits the Growth of Tumor Xenografts 1"

    Article Title: Monoclonal Antibody 16D10 to the C-Terminal Domain of the Feto-Acinar Pancreatic Protein Binds to Membrane of Human Pancreatic Tumoral SOJ-6 Cells and Inhibits the Growth of Tumor Xenografts 1

    Journal: Neoplasia (New York, N.Y.)

    doi:

    Immunodetection of biotinylated cell surface antigens in SOJ-6 cells. SOJ-6 cells were grown until confluence, and membrane proteins and peptides were biotinylated and lysed. Then biotinylated membrane proteins were purified on streptavidin-agarose affinity column. The material eluted with 0.1 M acetic acid was separated on SDS-PAGE and electrotransferred onto nitrocellulose membranes, which were finally probed with antibodies: antibiotin (lane 1; 0.5 µg of protein), pAbL64 (lane 2; 5 µg of protein), mAbJ28 (lane 3; 5 µg of protein), mAb16D10 (lane 4; 5 µg of protein), pAbAntipeptide (lane 5; 5 µg of protein), and antibodies to Glut-1 (lane 5; 5 µg of protein). Arrow indicates the apparent molecular mass of detected peptides.
    Figure Legend Snippet: Immunodetection of biotinylated cell surface antigens in SOJ-6 cells. SOJ-6 cells were grown until confluence, and membrane proteins and peptides were biotinylated and lysed. Then biotinylated membrane proteins were purified on streptavidin-agarose affinity column. The material eluted with 0.1 M acetic acid was separated on SDS-PAGE and electrotransferred onto nitrocellulose membranes, which were finally probed with antibodies: antibiotin (lane 1; 0.5 µg of protein), pAbL64 (lane 2; 5 µg of protein), mAbJ28 (lane 3; 5 µg of protein), mAb16D10 (lane 4; 5 µg of protein), pAbAntipeptide (lane 5; 5 µg of protein), and antibodies to Glut-1 (lane 5; 5 µg of protein). Arrow indicates the apparent molecular mass of detected peptides.

    Techniques Used: Immunodetection, Purification, Affinity Column, SDS Page

    7) Product Images from "The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii"

    Article Title: The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii

    Journal: Infection and Immunity

    doi:

    Approximately six dominant surface proteins, including Ncp29 and Ncp35, were revealed by surface biotinylation of N. caninum tachyzoites and Western blot detection with avidin-HRP conjugate. (A) Ncp29 (6C11) and Ncp35 (5H5) that were immunoprecipitated (IP) from labeled parasites were biotinylated, as determined by Western blotting with avidin-HRP. (B) Similarly, MAb 6C11 (α-p29) detected Ncp29 and MAb 5H5 (α-p35) detected Ncp35 in the biotinylated protein fraction precipitated with streptavidin-agarose (StAv) but not in the protein fraction from nonbiotinylated parasites, thus indicating that the streptavidin precipitation was specific for biotin-labeled proteins. Avidin-HRP detection was specific for biotinylated proteins, as revealed by Western blot analysis of nonbiotinylated parasites.
    Figure Legend Snippet: Approximately six dominant surface proteins, including Ncp29 and Ncp35, were revealed by surface biotinylation of N. caninum tachyzoites and Western blot detection with avidin-HRP conjugate. (A) Ncp29 (6C11) and Ncp35 (5H5) that were immunoprecipitated (IP) from labeled parasites were biotinylated, as determined by Western blotting with avidin-HRP. (B) Similarly, MAb 6C11 (α-p29) detected Ncp29 and MAb 5H5 (α-p35) detected Ncp35 in the biotinylated protein fraction precipitated with streptavidin-agarose (StAv) but not in the protein fraction from nonbiotinylated parasites, thus indicating that the streptavidin precipitation was specific for biotin-labeled proteins. Avidin-HRP detection was specific for biotinylated proteins, as revealed by Western blot analysis of nonbiotinylated parasites.

    Techniques Used: Western Blot, Avidin-Biotin Assay, Immunoprecipitation, Labeling

    8) Product Images from "INTRACELLULAR GLUTATHIONE MEDIATES THE DENITROSYLATION OF PROTEIN NITROSOTHIOLS IN THE RAT SPINAL CORD"

    Article Title: INTRACELLULAR GLUTATHIONE MEDIATES THE DENITROSYLATION OF PROTEIN NITROSOTHIOLS IN THE RAT SPINAL CORD

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.21897

    Rate of denitrosylation of specific S-nitrosoproteins. Rat SC slices were incubated with 1 mM GSNO plus 10 mM DEM for 2 h. Sections were then rinsed in fresh HBSS solution and the incubation continued for 3 h in the presence of 1 mM GSH-EE. After incubation, proteins were derivatized with HPDP-biotin and biotinylated proteins were isolated with streptavidin-agarose as described under “Materials and Methods”. Aliquots from the total (T) and bound (B) fractions were analyzed by western blotting using various monoclonal antibodies. Values are expressed as the percentage of protein that is S-nitrosated and represent the mean ± SEM of 3 separate experiments. *Significantly different ( p
    Figure Legend Snippet: Rate of denitrosylation of specific S-nitrosoproteins. Rat SC slices were incubated with 1 mM GSNO plus 10 mM DEM for 2 h. Sections were then rinsed in fresh HBSS solution and the incubation continued for 3 h in the presence of 1 mM GSH-EE. After incubation, proteins were derivatized with HPDP-biotin and biotinylated proteins were isolated with streptavidin-agarose as described under “Materials and Methods”. Aliquots from the total (T) and bound (B) fractions were analyzed by western blotting using various monoclonal antibodies. Values are expressed as the percentage of protein that is S-nitrosated and represent the mean ± SEM of 3 separate experiments. *Significantly different ( p

    Techniques Used: Incubation, Isolation, Western Blot

    9) Product Images from "Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs"

    Article Title: Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

    Journal: Viruses

    doi: 10.3390/v8020039

    Schematic of poliovirus genomic RNAs containing S1 and D8 aptamer tags within the highly structured 5′NCR, for positive-strand isolation. Insertion of either the S1 streptavidin-binding or D8 Sephadex-binding aptamer sequence within three regions of the poliovirus 5′NCR resulted in the production of 12 poliovirus cDNA constructs. Tag insertions in place of S-L III (blue), in place of S-L VI (green), or at nucleotide position 702 (purple) are depicted on the left, middle, and right, respectively. Constructs for negative-strand isolation (aptamers inserted in a reverse orientation) are not shown.
    Figure Legend Snippet: Schematic of poliovirus genomic RNAs containing S1 and D8 aptamer tags within the highly structured 5′NCR, for positive-strand isolation. Insertion of either the S1 streptavidin-binding or D8 Sephadex-binding aptamer sequence within three regions of the poliovirus 5′NCR resulted in the production of 12 poliovirus cDNA constructs. Tag insertions in place of S-L III (blue), in place of S-L VI (green), or at nucleotide position 702 (purple) are depicted on the left, middle, and right, respectively. Constructs for negative-strand isolation (aptamers inserted in a reverse orientation) are not shown.

    Techniques Used: Isolation, Binding Assay, Sequencing, Construct

    10) Product Images from "RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins"

    Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2016.12.014

    RhoA is a substrate for S-nitrosylation. (A) HLMVECs were treated with either vehicle or Cys-NO (100 μM) for 30 min, and the S-nitrosylation of proteins was determined by the biotin-switch assay in the presence of ascorbate and trace levels of copper. Biotinylated proteins were concentrated using streptavidin–agarose beads, and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (B) HLMVECs were treated with or without Cys-NO (100 μM) for 30 min, and S-nitrosylated proteins were selected using organomercury columns followed by immunoblotting for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). The relative densitometry of SNO-RhoA vs total-RhoA is expressed as means ± S.E., * P
    Figure Legend Snippet: RhoA is a substrate for S-nitrosylation. (A) HLMVECs were treated with either vehicle or Cys-NO (100 μM) for 30 min, and the S-nitrosylation of proteins was determined by the biotin-switch assay in the presence of ascorbate and trace levels of copper. Biotinylated proteins were concentrated using streptavidin–agarose beads, and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (B) HLMVECs were treated with or without Cys-NO (100 μM) for 30 min, and S-nitrosylated proteins were selected using organomercury columns followed by immunoblotting for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). The relative densitometry of SNO-RhoA vs total-RhoA is expressed as means ± S.E., * P

    Techniques Used: Biotin Switch Assay

    Mutation of RhoA on C16, 20, 159S reduces the eNOS-dependent S-nitrosylation of RhoA and protects RhoA from the inhibitory effects of NO. (A) COS-7 cells transfected with WT or mutant C16, 20, 159S RhoA constructs were treated with or without Cys-NO (100 μM) for 30 min. Cells were then lysed, the biotin-switch assay performed and biotinylated proteins concentrated using streptavidin agarose. Total S-nitrosylated proteins were identified using an anti-biotin antibody (top panel) and S-nitrosylated RhoA using a RhoA antibody (lower panel). (B) HEK293-eNOS cells were transfected with RhoA WT or the RhoAC16, 20, 159S mutant, and the degree of S-nitrosylation of RhoA was determined using the biotin-switch assay and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (C) COS-7 cells were transfected with WT or mutant C16, 20, 159S RhoA and exposed to the indicated concentrations of Cys-NO for 30 min. Cells were then lysed and RhoA activity determined using the G-LISA RhoA activation assay. Data are expressed as means ± S.E., * P
    Figure Legend Snippet: Mutation of RhoA on C16, 20, 159S reduces the eNOS-dependent S-nitrosylation of RhoA and protects RhoA from the inhibitory effects of NO. (A) COS-7 cells transfected with WT or mutant C16, 20, 159S RhoA constructs were treated with or without Cys-NO (100 μM) for 30 min. Cells were then lysed, the biotin-switch assay performed and biotinylated proteins concentrated using streptavidin agarose. Total S-nitrosylated proteins were identified using an anti-biotin antibody (top panel) and S-nitrosylated RhoA using a RhoA antibody (lower panel). (B) HEK293-eNOS cells were transfected with RhoA WT or the RhoAC16, 20, 159S mutant, and the degree of S-nitrosylation of RhoA was determined using the biotin-switch assay and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (C) COS-7 cells were transfected with WT or mutant C16, 20, 159S RhoA and exposed to the indicated concentrations of Cys-NO for 30 min. Cells were then lysed and RhoA activity determined using the G-LISA RhoA activation assay. Data are expressed as means ± S.E., * P

    Techniques Used: Mutagenesis, Transfection, Construct, Biotin Switch Assay, Activity Assay, Activation Assay

    11) Product Images from "Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *"

    Article Title: Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor ?B (NF-?B) Proteins *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.451153

    DNA binding profiles of Nematostella and human NF-κB dimers. A , NF-κB proteins were expressed in E. coli , and the soluble fractions of bacterial lysates ( Input ; separated from insoluble debris by centrifugation) were incubated for 2 h at room temperature with streptavidin-agarose attached to biotinylated DNA oligonucleotides containing NF-κB-binding (from the 3′-region of the TNF gene) or control sequences. The bound proteins were eluted with high-salt buffer, and the eluates were subjected to SDS-PAGE, followed by Coomassie Blue staining. B , a heat map of the binding profiles based on the microarray analysis of four NF-κB dimers (presented in columns ) was generated using MultiExperiment Viewer ( 25 , 26 ). The z -scores ( supplemental Table 1 ) of each individual protein were used as input for MultiExperiment Viewer. Within the heat map, probes that contain the 803 11-mer sequences and represent k-mer space given by the consensus sequence RGGRNNHHYYB can be found as rows . A color gradient reflects the binding affinity z -scores of NF-κB dimers for a probe, where high-affinity probes (positive values) are shown in yellow , z -scores near zero are shown in black , and low-affinity probes (negative values) are shown in blue (see side bar ). Hierarchical clustering was used to describe relationships between binding profiles of the different dimers (Euclidean distance correlation and complete linkage analysis). C , pairwise comparisons of the DNA binding profiles of Nematostella and human NF-κB dimers based on the array analysis. The value at the bottom of each graph is the correlation coefficient for the pair in the graph. These graphs were built for the z -score data sets. z -scores were obtained using log 2 -transformed intensities, and the median of replicates were calculated for each probe within every array. D , DNA barcodes of the NF-κB proteins based on the top 20 highest binding motifs for each individual protein were created using WebLogo online software. E , recruitment of human and Nematostella NF-κB p50 to human gene promoters. Plasmids encoding Myc-tagged HsNF-κB p50 and NvNF-κB p50 proteins were transfected into human 293ET cells. One day after transfection, cells were lysed, and the lysates were subjected to ChIP using anti-Myc (clone 9E10) or IgG control antibodies. The NF-κB recruitment to gene promoters was analyzed by qPCR of the precipitated DNA using specific primers to the human TNF and IL-10 gene promoters. The data are presented as -fold change over a negative IgG control.
    Figure Legend Snippet: DNA binding profiles of Nematostella and human NF-κB dimers. A , NF-κB proteins were expressed in E. coli , and the soluble fractions of bacterial lysates ( Input ; separated from insoluble debris by centrifugation) were incubated for 2 h at room temperature with streptavidin-agarose attached to biotinylated DNA oligonucleotides containing NF-κB-binding (from the 3′-region of the TNF gene) or control sequences. The bound proteins were eluted with high-salt buffer, and the eluates were subjected to SDS-PAGE, followed by Coomassie Blue staining. B , a heat map of the binding profiles based on the microarray analysis of four NF-κB dimers (presented in columns ) was generated using MultiExperiment Viewer ( 25 , 26 ). The z -scores ( supplemental Table 1 ) of each individual protein were used as input for MultiExperiment Viewer. Within the heat map, probes that contain the 803 11-mer sequences and represent k-mer space given by the consensus sequence RGGRNNHHYYB can be found as rows . A color gradient reflects the binding affinity z -scores of NF-κB dimers for a probe, where high-affinity probes (positive values) are shown in yellow , z -scores near zero are shown in black , and low-affinity probes (negative values) are shown in blue (see side bar ). Hierarchical clustering was used to describe relationships between binding profiles of the different dimers (Euclidean distance correlation and complete linkage analysis). C , pairwise comparisons of the DNA binding profiles of Nematostella and human NF-κB dimers based on the array analysis. The value at the bottom of each graph is the correlation coefficient for the pair in the graph. These graphs were built for the z -score data sets. z -scores were obtained using log 2 -transformed intensities, and the median of replicates were calculated for each probe within every array. D , DNA barcodes of the NF-κB proteins based on the top 20 highest binding motifs for each individual protein were created using WebLogo online software. E , recruitment of human and Nematostella NF-κB p50 to human gene promoters. Plasmids encoding Myc-tagged HsNF-κB p50 and NvNF-κB p50 proteins were transfected into human 293ET cells. One day after transfection, cells were lysed, and the lysates were subjected to ChIP using anti-Myc (clone 9E10) or IgG control antibodies. The NF-κB recruitment to gene promoters was analyzed by qPCR of the precipitated DNA using specific primers to the human TNF and IL-10 gene promoters. The data are presented as -fold change over a negative IgG control.

    Techniques Used: Binding Assay, Centrifugation, Incubation, SDS Page, Staining, Microarray, Generated, Sequencing, Transformation Assay, Software, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    12) Product Images from "Ubiquitylated H2A.Z nucleosomes are associated with nuclear architectural proteins and global transcriptional silencing"

    Article Title: Ubiquitylated H2A.Z nucleosomes are associated with nuclear architectural proteins and global transcriptional silencing

    Journal: bioRxiv

    doi: 10.1101/759852

    Separation of distinct pools of H2A.Z-FB nucleosomes by streptavidin (SA) or Flag antibody-immunoprecipitation. (A) Cartoon diagram showing the expected types of H2A.Z-FB nucleosomes found in the H2A.Z-FB + Avi-Ub transfected cells and the distinct types of nucleosomes pulled down by SA or Flag antibody immunoprecipitation. (B) Coomassie stained gel of the input mono-nucleosome fraction and SA- or Flag antibody-immunoprecipitated nucleosomes. * indicates non-specific bands often seen in SA-IPs (C) Western blot analysis of the input and immunoprecipitated nucleosomes using Flag antibody and Avidin-HRP. The different types of ubiquitylated and non-ubiquitylated H2A.Z-FB (illustrated on the right hand side and marked by single, double and triple asterisks) were resolved on long SDS polyacrylamide gels and detected by Flag antibody.
    Figure Legend Snippet: Separation of distinct pools of H2A.Z-FB nucleosomes by streptavidin (SA) or Flag antibody-immunoprecipitation. (A) Cartoon diagram showing the expected types of H2A.Z-FB nucleosomes found in the H2A.Z-FB + Avi-Ub transfected cells and the distinct types of nucleosomes pulled down by SA or Flag antibody immunoprecipitation. (B) Coomassie stained gel of the input mono-nucleosome fraction and SA- or Flag antibody-immunoprecipitated nucleosomes. * indicates non-specific bands often seen in SA-IPs (C) Western blot analysis of the input and immunoprecipitated nucleosomes using Flag antibody and Avidin-HRP. The different types of ubiquitylated and non-ubiquitylated H2A.Z-FB (illustrated on the right hand side and marked by single, double and triple asterisks) were resolved on long SDS polyacrylamide gels and detected by Flag antibody.

    Techniques Used: Immunoprecipitation, Transfection, Staining, Western Blot, Avidin-Biotin Assay

    13) Product Images from "Interaction of fascin and protein kinase C?: a novel intersection in cell adhesion and motility"

    Article Title: Interaction of fascin and protein kinase C?: a novel intersection in cell adhesion and motility

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdg521

    Fig. 4. Biochemical properties of TAT-FAS peptides. ( A ) Design and amino acid sequences of TAT-FAS peptides. ( B ) Binding specificity of FAS-TAT peptides for actin. Pull-downs were performed with the different biotinylated TAT-FAS peptides bound to streptavidin–agarose as indicated. Candidate partner proteins were detected by immunoblot of SDS–PAGE gels. For each sample in (i) and (ii), the whole of the bound fraction and one-fifth of the supernatant (SN) was loaded per lane. (i) Binding to purified F-actin; (ii) binding specificity for actin in cell lysates; and (iii) competition of actin binding to purified fascin-His by free peptides. For each peptide: lane 1, 100 nM; lane 2, 300 nM; lane 3, 600 nM free peptide. Beads, Ni-NTA without fascin; actin +ve, cell extract. ( C ) Binding specificities of TAT-FAS peptides for PKCα. (i) Pull-down assays with TAT-FAS peptides bound to streptavidin–agarose as indicated were carried out on lysates of MCF-7 cells expressing GFP–PKCα-myc and immunoblotted for PKCα. Samples were reprobed for α-tubulin to demonstrate binding specificity. (ii) Cross-competition for PKCα binding to TAT–fascin S39D peptide–beads by free peptides. Cell extracts were preincubated with free TAT–fascin peptides as indicated for 1 h at 4°C before addition of TAT-FAS S39D–beads. For each sample, the whole of the bound fraction (B) and one-fifth of the supernatant (SN) was loaded per lane. ( D ) (i) Direct binding of fascin to active PKCα. Ni-NTA beads were left unloaded or loaded with 400 ng of purified recombinant fascin, washed, and incubated with 100 ng recombinant PKCα in the absence or presence of activating lipids and free TAT-FAS peptides as indicated. For each peptide: lane 1, 100 nM; lane 2, 300 nM; lane 3, 600 nM free peptide. (ii) Competition of fascin coimmunoprecipitation with PKCα by TAT-FAS S39D. C2C12 cells were preloaded with 300 nM TAT-FAS peptides as indicated, plated on 50 nM FN for 1 h, then immunoprecipitated for PKCα. Associated fascin was detected by immunoblot.
    Figure Legend Snippet: Fig. 4. Biochemical properties of TAT-FAS peptides. ( A ) Design and amino acid sequences of TAT-FAS peptides. ( B ) Binding specificity of FAS-TAT peptides for actin. Pull-downs were performed with the different biotinylated TAT-FAS peptides bound to streptavidin–agarose as indicated. Candidate partner proteins were detected by immunoblot of SDS–PAGE gels. For each sample in (i) and (ii), the whole of the bound fraction and one-fifth of the supernatant (SN) was loaded per lane. (i) Binding to purified F-actin; (ii) binding specificity for actin in cell lysates; and (iii) competition of actin binding to purified fascin-His by free peptides. For each peptide: lane 1, 100 nM; lane 2, 300 nM; lane 3, 600 nM free peptide. Beads, Ni-NTA without fascin; actin +ve, cell extract. ( C ) Binding specificities of TAT-FAS peptides for PKCα. (i) Pull-down assays with TAT-FAS peptides bound to streptavidin–agarose as indicated were carried out on lysates of MCF-7 cells expressing GFP–PKCα-myc and immunoblotted for PKCα. Samples were reprobed for α-tubulin to demonstrate binding specificity. (ii) Cross-competition for PKCα binding to TAT–fascin S39D peptide–beads by free peptides. Cell extracts were preincubated with free TAT–fascin peptides as indicated for 1 h at 4°C before addition of TAT-FAS S39D–beads. For each sample, the whole of the bound fraction (B) and one-fifth of the supernatant (SN) was loaded per lane. ( D ) (i) Direct binding of fascin to active PKCα. Ni-NTA beads were left unloaded or loaded with 400 ng of purified recombinant fascin, washed, and incubated with 100 ng recombinant PKCα in the absence or presence of activating lipids and free TAT-FAS peptides as indicated. For each peptide: lane 1, 100 nM; lane 2, 300 nM; lane 3, 600 nM free peptide. (ii) Competition of fascin coimmunoprecipitation with PKCα by TAT-FAS S39D. C2C12 cells were preloaded with 300 nM TAT-FAS peptides as indicated, plated on 50 nM FN for 1 h, then immunoprecipitated for PKCα. Associated fascin was detected by immunoblot.

    Techniques Used: Binding Assay, SDS Page, Purification, Expressing, Recombinant, Incubation, Immunoprecipitation

    14) Product Images from "Interaction of Muscle and Brain Sodium Channels with Multiple Members of the Syntrophin Family of Dystrophin-Associated Proteins"

    Article Title: Interaction of Muscle and Brain Sodium Channels with Multiple Members of the Syntrophin Family of Dystrophin-Associated Proteins

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.18-01-00128.1998

    The C termini of muscle NaChs bind directly to syntrophin PDZ domains. A , Biotinylated peptides corresponding to the C-terminal 10 amino acids of the SkM1 and SkM2 NaChs or Fas bound to streptavidin–HRP were overlaid onto α1-syntrophin
    Figure Legend Snippet: The C termini of muscle NaChs bind directly to syntrophin PDZ domains. A , Biotinylated peptides corresponding to the C-terminal 10 amino acids of the SkM1 and SkM2 NaChs or Fas bound to streptavidin–HRP were overlaid onto α1-syntrophin

    Techniques Used:

    15) Product Images from "A Unique Element in the Cytoplasmic Tail of the Type II Transforming Growth Factor-? Receptor Controls Basolateral Delivery"

    Article Title: A Unique Element in the Cytoplasmic Tail of the Type II Transforming Growth Factor-? Receptor Controls Basolateral Delivery

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E06-10-0930

    Biotin T2R labeling at apical or basolateral membrane domains. Stable MDCK cell lines expressing wild type (A), A531G point-mutated (B) or βIIΔ537-truncated (C) βII chimeric receptor constructs were biotin labeled apically (a) or basolaterally (b) as polarized monolayers or as a nonpolarized monolayer for total labeling (t). Biotinylated protein was extracted on streptavidin-agarose beads and Western blotted using a receptor specific antibody. Bottom, control nonlabeled cells are designated c. Top, XZ cross sections of parallel immunofluorescently stained polarized monolayers.
    Figure Legend Snippet: Biotin T2R labeling at apical or basolateral membrane domains. Stable MDCK cell lines expressing wild type (A), A531G point-mutated (B) or βIIΔ537-truncated (C) βII chimeric receptor constructs were biotin labeled apically (a) or basolaterally (b) as polarized monolayers or as a nonpolarized monolayer for total labeling (t). Biotinylated protein was extracted on streptavidin-agarose beads and Western blotted using a receptor specific antibody. Bottom, control nonlabeled cells are designated c. Top, XZ cross sections of parallel immunofluorescently stained polarized monolayers.

    Techniques Used: Labeling, Expressing, Construct, Western Blot, Staining

    16) Product Images from "Ubiquitin-dependent folding of the Wnt signaling coreceptor LRP6"

    Article Title: Ubiquitin-dependent folding of the Wnt signaling coreceptor LRP6

    Journal: eLife

    doi: 10.7554/eLife.19083

    usp19 silencing leads to decrease in LRP6 cell surface expression. Surface Biotinylation assay performed in RPE1 cells upon 24, 48 and 72 hr of usp19 or usp13 gene silencing. Quantification of endogenous LRP6 surface expression at 48 hr of gene silencing in Streptavidin-mediated pull down is shown above the western blot. Errors represent standard deviation (n = 3) and ***
    Figure Legend Snippet: usp19 silencing leads to decrease in LRP6 cell surface expression. Surface Biotinylation assay performed in RPE1 cells upon 24, 48 and 72 hr of usp19 or usp13 gene silencing. Quantification of endogenous LRP6 surface expression at 48 hr of gene silencing in Streptavidin-mediated pull down is shown above the western blot. Errors represent standard deviation (n = 3) and ***

    Techniques Used: Expressing, Surface Biotinylation Assay, Western Blot, Standard Deviation

    17) Product Images from "Peptide EphB2/CTF2 Generated by the ?-Secretase Processing of EphB2 Receptor Promotes Tyrosine Phosphorylation and Cell Surface Localization of N-Methyl-d-aspartate Receptors *"

    Article Title: Peptide EphB2/CTF2 Generated by the ?-Secretase Processing of EphB2 Receptor Promotes Tyrosine Phosphorylation and Cell Surface Localization of N-Methyl-d-aspartate Receptors *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.048728

    EphB2/CTF2 increases surface expression of NMDAR. A , HEK293T cells were transfected with rEphB2/CTF2, rEphB2/CTF2-K664M ( KD ), or vector ( V ) along with NR1 and NR2B as indicated. The cell surface proteins were labeled with biotin, purified with streptavidin,
    Figure Legend Snippet: EphB2/CTF2 increases surface expression of NMDAR. A , HEK293T cells were transfected with rEphB2/CTF2, rEphB2/CTF2-K664M ( KD ), or vector ( V ) along with NR1 and NR2B as indicated. The cell surface proteins were labeled with biotin, purified with streptavidin,

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Labeling, Purification

    18) Product Images from "Herp Regulates Hrd1-mediated Ubiquitylation in a Ubiquitin-like Domain-dependent Manner *"

    Article Title: Herp Regulates Hrd1-mediated Ubiquitylation in a Ubiquitin-like Domain-dependent Manner *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.134551

    Oligomerization of Hrd1. A , HeLa cells ( ctrl ) and Hrd1-HTB cells ( clone 6 and clone 36 ) were lysed and subjected either to immunoprecipitation using Hrd1 antiserum ( Hrd1 IP ) or to streptavidin-agarose pull-down ( strept. PD Hrd1-HTB ). GAPDH was used as a control, demonstrating similar protein concentrations in the cell extracts. Precipitates were analyzed by Western blotting as indicated. A dash indicates the position of a 72-kDa protein marker, one asterisk indicates the position of WT-Herp, and two asterisks indicate the position of Herp-HTB. short exp. , short exposure; long exp. , long exposure. B , Hrd1-HTB cell extracts (clone 36) were subjected to glycerol gradient centrifugation. Fractions were incubated with streptavidin-agarose to precipitate Hrd1-HTB-containing complexes, which were analyzed by immunoblotting as indicated. For comparison, sedimentation of PA28 (∼200 kDa) and proteasomes (∼700–3000 kDa) is indicated by bars labeled accordingly. C , Hrd1-HTB cells (clone 36) were treated with 50 μg/ml CHX for the indicated periods of time. Cell extracts ( extr. ) were subjected to streptavidin-agarose pull-down ( strept. PD ). Precipitates were analyzed by immunoblotting as indicated. GAPDH was used as a loading control.
    Figure Legend Snippet: Oligomerization of Hrd1. A , HeLa cells ( ctrl ) and Hrd1-HTB cells ( clone 6 and clone 36 ) were lysed and subjected either to immunoprecipitation using Hrd1 antiserum ( Hrd1 IP ) or to streptavidin-agarose pull-down ( strept. PD Hrd1-HTB ). GAPDH was used as a control, demonstrating similar protein concentrations in the cell extracts. Precipitates were analyzed by Western blotting as indicated. A dash indicates the position of a 72-kDa protein marker, one asterisk indicates the position of WT-Herp, and two asterisks indicate the position of Herp-HTB. short exp. , short exposure; long exp. , long exposure. B , Hrd1-HTB cell extracts (clone 36) were subjected to glycerol gradient centrifugation. Fractions were incubated with streptavidin-agarose to precipitate Hrd1-HTB-containing complexes, which were analyzed by immunoblotting as indicated. For comparison, sedimentation of PA28 (∼200 kDa) and proteasomes (∼700–3000 kDa) is indicated by bars labeled accordingly. C , Hrd1-HTB cells (clone 36) were treated with 50 μg/ml CHX for the indicated periods of time. Cell extracts ( extr. ) were subjected to streptavidin-agarose pull-down ( strept. PD ). Precipitates were analyzed by immunoblotting as indicated. GAPDH was used as a loading control.

    Techniques Used: Immunoprecipitation, Western Blot, Marker, Gradient Centrifugation, Incubation, Sedimentation, Labeling

    19) Product Images from "Interactome Analyses Identify Ties of PrPC and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones"

    Article Title: Interactome Analyses Identify Ties of PrPC and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000608

    Evidence for cell surface localization of a subset of PDIs and calreticulin in mouse neuroblastoma cells. N2a and ScN2a cells were subjected to cell surface biotinylation or mock treatment. Subsequently, cellular extracts were side-by-side affinity purified on a streptavidin agarose matrix. Extracts and streptavidin agarose eluate fractions were analyzed by Western blotting and membranes probed with antibodies directed against histone 2B, PrP, P4hb, Pdia3 and calreticulin. The relative strength of P4hb-, Pdia3- and calreticulin-specific signals present in eluate fractions from biotinylated (lanes 6 and 8) versus non-biotinylated samples (lanes 5 and 7) is consistent with the conclusion that a subset of these proteins resided at the cell surface during the biotinylation step. The relative intensity of P4hb- and Pdia3-derived signals in extract (40 µg total protein loaded per lane) and eluate fractions can be estimated from the concomitant analysis of 1/4 and 1/20 of the extract fraction shown in lane 1. Thus, P4hb and Pdia3 signals in the eluate fraction (captured from 2 mg total extract) were equivalent to or exceeded the amount of these proteins present in 2 µg of extract. Please note the presence of histone 2B in all eluate fractions (lanes 5–8), in support of the conclusion that the protein binds unspecifically to the affinity matrix.
    Figure Legend Snippet: Evidence for cell surface localization of a subset of PDIs and calreticulin in mouse neuroblastoma cells. N2a and ScN2a cells were subjected to cell surface biotinylation or mock treatment. Subsequently, cellular extracts were side-by-side affinity purified on a streptavidin agarose matrix. Extracts and streptavidin agarose eluate fractions were analyzed by Western blotting and membranes probed with antibodies directed against histone 2B, PrP, P4hb, Pdia3 and calreticulin. The relative strength of P4hb-, Pdia3- and calreticulin-specific signals present in eluate fractions from biotinylated (lanes 6 and 8) versus non-biotinylated samples (lanes 5 and 7) is consistent with the conclusion that a subset of these proteins resided at the cell surface during the biotinylation step. The relative intensity of P4hb- and Pdia3-derived signals in extract (40 µg total protein loaded per lane) and eluate fractions can be estimated from the concomitant analysis of 1/4 and 1/20 of the extract fraction shown in lane 1. Thus, P4hb and Pdia3 signals in the eluate fraction (captured from 2 mg total extract) were equivalent to or exceeded the amount of these proteins present in 2 µg of extract. Please note the presence of histone 2B in all eluate fractions (lanes 5–8), in support of the conclusion that the protein binds unspecifically to the affinity matrix.

    Techniques Used: Affinity Purification, Western Blot, Derivative Assay

    20) Product Images from "Dynamic Interplay between Adhesive and Lateral E-Cadherin Dimers"

    Article Title: Dynamic Interplay between Adhesive and Lateral E-Cadherin Dimers

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.21.7449-7458.2002

    Fast incorporation of 35 S-labeled E-cadherin into dimers. A-431 cells were biotinylated and then pulse-labeled with [ 35 S]methionine/cysteine. Next, the cells were chased for 15, 30, 60, or 180 min (chase times in minutes are indicated above the blots). After extraction and precipitation by streptavidin-biotin agarose, proteins were eluted from the beads and then immunoprecipitated with an E-cadherin-specific antibody. The autoradiogram ( 35 S) of the immunoprecipitates shows that the incorporation of the 35 S-labeled E-cadherin into dimers containing a biotinylated form of the same protein reached a plateau after 30 min. Staining the same blot with E-cadherin antibody (Ec) demonstrated that all precipitates contained the same amounts of E-cadherin. In the control experiment (lane C), lysates obtained from biotinylated and metabolically labeled cells (after 30-min chase), were mixed and processed as described above. No E-cadherin-derived signal was detected in the autoradiogram.
    Figure Legend Snippet: Fast incorporation of 35 S-labeled E-cadherin into dimers. A-431 cells were biotinylated and then pulse-labeled with [ 35 S]methionine/cysteine. Next, the cells were chased for 15, 30, 60, or 180 min (chase times in minutes are indicated above the blots). After extraction and precipitation by streptavidin-biotin agarose, proteins were eluted from the beads and then immunoprecipitated with an E-cadherin-specific antibody. The autoradiogram ( 35 S) of the immunoprecipitates shows that the incorporation of the 35 S-labeled E-cadherin into dimers containing a biotinylated form of the same protein reached a plateau after 30 min. Staining the same blot with E-cadherin antibody (Ec) demonstrated that all precipitates contained the same amounts of E-cadherin. In the control experiment (lane C), lysates obtained from biotinylated and metabolically labeled cells (after 30-min chase), were mixed and processed as described above. No E-cadherin-derived signal was detected in the autoradiogram.

    Techniques Used: Labeling, Immunoprecipitation, Staining, Metabolic Labelling, Derivative Assay

    Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with streptavidin-HRP (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.
    Figure Legend Snippet: Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with streptavidin-HRP (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.

    Techniques Used: Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Gradient Centrifugation, Lysis

    21) Product Images from "Immunodeficiency, auto-inflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency"

    Article Title: Immunodeficiency, auto-inflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency

    Journal: Nature immunology

    doi: 10.1038/ni.2457

    Impaired recruitment of NEMO to cytokine receptors in the patients’ fibroblasts a) HOIL-1 deficiency impairs the recruitment of NEMO to the TNF-RSC. Flag-tagged TNF was used to activate and isolate the TNF-RSC. Cells were lysed, TNF-RSC was purified on Flag-affinity resin and complex-associated NEMO was analyzed by immunoblotting. b) HOIL-1 deficiency abolishes NEMO recruitment to the IL-1-RSC. The same strategy as in a) was used, except that Flag-tagged IL-1β was used to stimulate the cells and to isolate the IL-1-RSC. The abundance of TNFR (a) and IL-1R (b), assessed by immunoblotting, was similar in control and patient-derived fibroblasts. c ) HOIL-1 deficiency impairs the interaction between NEMO and polyubiquitinated RIP1 in response to TNF. NEMO (NEMO-IP) was immunoprecipitated from lysates of control and patient fibroblasts treated with biotinylated TNF and analyzed by immunoblotting for NEMO, RIP1, and IKKβ. The total amount of the ubiquitinated forms of RIP1 associated with the TNF-RSC was evaluated by streptavidin pulldown followed by immunoblotting for RIP1. d ) HOIL-1 deficiency impairs the interaction between NEMO and polyubiquitinated IRAK-1 in response to IL-1β. NEMO and IRAK-1 were immunoprecipitated from lysates of IL-1β-treated fibroblasts and subjected to western blotting for NEMO or IRAK-1 as indicated. These data are representative of three experiments.
    Figure Legend Snippet: Impaired recruitment of NEMO to cytokine receptors in the patients’ fibroblasts a) HOIL-1 deficiency impairs the recruitment of NEMO to the TNF-RSC. Flag-tagged TNF was used to activate and isolate the TNF-RSC. Cells were lysed, TNF-RSC was purified on Flag-affinity resin and complex-associated NEMO was analyzed by immunoblotting. b) HOIL-1 deficiency abolishes NEMO recruitment to the IL-1-RSC. The same strategy as in a) was used, except that Flag-tagged IL-1β was used to stimulate the cells and to isolate the IL-1-RSC. The abundance of TNFR (a) and IL-1R (b), assessed by immunoblotting, was similar in control and patient-derived fibroblasts. c ) HOIL-1 deficiency impairs the interaction between NEMO and polyubiquitinated RIP1 in response to TNF. NEMO (NEMO-IP) was immunoprecipitated from lysates of control and patient fibroblasts treated with biotinylated TNF and analyzed by immunoblotting for NEMO, RIP1, and IKKβ. The total amount of the ubiquitinated forms of RIP1 associated with the TNF-RSC was evaluated by streptavidin pulldown followed by immunoblotting for RIP1. d ) HOIL-1 deficiency impairs the interaction between NEMO and polyubiquitinated IRAK-1 in response to IL-1β. NEMO and IRAK-1 were immunoprecipitated from lysates of IL-1β-treated fibroblasts and subjected to western blotting for NEMO or IRAK-1 as indicated. These data are representative of three experiments.

    Techniques Used: Purification, Derivative Assay, Immunoprecipitation, Western Blot

    22) Product Images from "An engineered antibody fragment targeting mutant β-catenin via major histocompatibility complex I neoantigen presentation"

    Article Title: An engineered antibody fragment targeting mutant β-catenin via major histocompatibility complex I neoantigen presentation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.010251

    Identification of S45F mutant β-catenin 41–49 -HLA-A*03:01–specific phage clones. A , five phage clones bind specifically to the β-catenin mutant pHLA-A3 by ELISA. pHLA-A3 complexes or streptavidin bound to plates were incubated with dilutions of the various phage clones (1:500, 1:2,500, and 1:12,500 from darkest to lightest in each color series), followed by detection with an anti-M13 antibody. Data are represented as mean ± S.D. ( error bars ). B , five phage clones bind specifically to mutant pHLA-A3 on the cell surface. T2A3 cells were peptide-pulsed with β 2 -microglobulin in the presence or absence of the specified peptide and then stained with the various phage clones and analyzed by flow cytometry. No significant binding was detected to any of the seven Blast peptides.
    Figure Legend Snippet: Identification of S45F mutant β-catenin 41–49 -HLA-A*03:01–specific phage clones. A , five phage clones bind specifically to the β-catenin mutant pHLA-A3 by ELISA. pHLA-A3 complexes or streptavidin bound to plates were incubated with dilutions of the various phage clones (1:500, 1:2,500, and 1:12,500 from darkest to lightest in each color series), followed by detection with an anti-M13 antibody. Data are represented as mean ± S.D. ( error bars ). B , five phage clones bind specifically to mutant pHLA-A3 on the cell surface. T2A3 cells were peptide-pulsed with β 2 -microglobulin in the presence or absence of the specified peptide and then stained with the various phage clones and analyzed by flow cytometry. No significant binding was detected to any of the seven Blast peptides.

    Techniques Used: Mutagenesis, Clone Assay, Enzyme-linked Immunosorbent Assay, Incubation, Staining, Flow Cytometry, Cytometry, Binding Assay

    Evaluation of scFv binding to S45F mutant and WT β-catenin 41–49 -HLA-A*03:01. A , analysis of recombinant scFv binding to mutant pHLA by ELISA. pHLA-A3 complexes or streptavidin bound to plates were incubated with decreasing concentration of scFvs (200, 40, 8, 1.6, and 0.64 n m from darkest to lightest in each color series). scFv binding was detected with protein L followed by an anti-protein L antibody. Data are represented as the average of three technical replicates ± S.D. ( error bars ). B , analysis of recombinant scFv binding to peptide-pulsed cells by flow cytometry. T2A3 cells were peptide-pulsed with β 2 -microglobulin and the specified peptide, stained with the various scFvs, and analyzed by flow cytometry. C–E , scFv binding to mutant and WT pHLA-A3 was evaluated by single-cycle kinetics using SPR. The scFv was loaded at increasing concentrations from 6.25 to 25, 100, and 400 n m for cl. 7 and cl. 3 and 15.6, 62.5, 250, and 1000 n m for E10. Blank- and reference-subtracted binding responses are shown in orange for the mutant pHLA-A3 and blue for the WT pHLA-A3; the fitted curve is shown in black or gray. C , cl. 7 displays one-to-one binding to the mutant pHLA-A3 and minimal binding to the WT pHLA-A3. D , E10 displays one-to-one binding to the mutant pHLA-A3 and minimal binding to the WT pHLA-A3. E , cl. 3 displays heterogeneous ligand binding to the mutant pHLA-A3 and displays some off-target binding to the WT pHLA-A3 at 100 and 400 n m . F , evaluation of cl. 7 and E10 binding to the mutant and WT peptides in complex with HLA-A*03:01 and HLA-A*11:01 by ELISA. Data are represented as the average of three technical replicates ± S.D.
    Figure Legend Snippet: Evaluation of scFv binding to S45F mutant and WT β-catenin 41–49 -HLA-A*03:01. A , analysis of recombinant scFv binding to mutant pHLA by ELISA. pHLA-A3 complexes or streptavidin bound to plates were incubated with decreasing concentration of scFvs (200, 40, 8, 1.6, and 0.64 n m from darkest to lightest in each color series). scFv binding was detected with protein L followed by an anti-protein L antibody. Data are represented as the average of three technical replicates ± S.D. ( error bars ). B , analysis of recombinant scFv binding to peptide-pulsed cells by flow cytometry. T2A3 cells were peptide-pulsed with β 2 -microglobulin and the specified peptide, stained with the various scFvs, and analyzed by flow cytometry. C–E , scFv binding to mutant and WT pHLA-A3 was evaluated by single-cycle kinetics using SPR. The scFv was loaded at increasing concentrations from 6.25 to 25, 100, and 400 n m for cl. 7 and cl. 3 and 15.6, 62.5, 250, and 1000 n m for E10. Blank- and reference-subtracted binding responses are shown in orange for the mutant pHLA-A3 and blue for the WT pHLA-A3; the fitted curve is shown in black or gray. C , cl. 7 displays one-to-one binding to the mutant pHLA-A3 and minimal binding to the WT pHLA-A3. D , E10 displays one-to-one binding to the mutant pHLA-A3 and minimal binding to the WT pHLA-A3. E , cl. 3 displays heterogeneous ligand binding to the mutant pHLA-A3 and displays some off-target binding to the WT pHLA-A3 at 100 and 400 n m . F , evaluation of cl. 7 and E10 binding to the mutant and WT peptides in complex with HLA-A*03:01 and HLA-A*11:01 by ELISA. Data are represented as the average of three technical replicates ± S.D.

    Techniques Used: Binding Assay, Mutagenesis, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Flow Cytometry, Cytometry, Staining, SPR Assay, Ligand Binding Assay

    23) Product Images from "Redox Proteomics of the Inflammatory Secretome Identifies a Common Set of Redoxins and Other Glutathionylated Proteins Released in Inflammation, Influenza Virus Infection and Oxidative Stress"

    Article Title: Redox Proteomics of the Inflammatory Secretome Identifies a Common Set of Redoxins and Other Glutathionylated Proteins Released in Inflammation, Influenza Virus Infection and Oxidative Stress

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127086

    Proteins released in glutathionylated form. Proteins in the NEM-blocked supernatants from BioGEE-pretreated, LPS-stimulated cells were immunoprecipitated with anti-PRDX1 (A) or anti-TXN1 (B). Immunoprecipitated proteins were run under non-reducing (two lanes on the left) or reducing conditions (the two lanes with DTT, on the right). Proteins were then visualized by Western blot with streptavidin peroxidase. The same blot was stripped and reprobed with anti-PRDX1 or anti-TXN1 antibody to locate the proteins (left, in both A and B). m, monomer; d, dimer.
    Figure Legend Snippet: Proteins released in glutathionylated form. Proteins in the NEM-blocked supernatants from BioGEE-pretreated, LPS-stimulated cells were immunoprecipitated with anti-PRDX1 (A) or anti-TXN1 (B). Immunoprecipitated proteins were run under non-reducing (two lanes on the left) or reducing conditions (the two lanes with DTT, on the right). Proteins were then visualized by Western blot with streptavidin peroxidase. The same blot was stripped and reprobed with anti-PRDX1 or anti-TXN1 antibody to locate the proteins (left, in both A and B). m, monomer; d, dimer.

    Techniques Used: Immunoprecipitation, Western Blot

    24) Product Images from "A protein complex required for polymerase V transcripts and RNA-directed DNA methylation in plants"

    Article Title: A protein complex required for polymerase V transcripts and RNA-directed DNA methylation in plants

    Journal: Current biology : CB

    doi: 10.1016/j.cub.2010.03.062

    Characterization of DDR complex components. (A–D) Streptavidin (SA) pulldown and co-immunopurification assays confirming interactions from Mass Spectrometric analyses. The BLRP tag is biotinylated in vivo allowing interaction with streptavidin.
    Figure Legend Snippet: Characterization of DDR complex components. (A–D) Streptavidin (SA) pulldown and co-immunopurification assays confirming interactions from Mass Spectrometric analyses. The BLRP tag is biotinylated in vivo allowing interaction with streptavidin.

    Techniques Used: Immu-Puri, In Vivo

    25) Product Images from "A protein complex required for polymerase V transcripts and RNA-directed DNA methylation in plants"

    Article Title: A protein complex required for polymerase V transcripts and RNA-directed DNA methylation in plants

    Journal: Current biology : CB

    doi: 10.1016/j.cub.2010.03.062

    Characterization of DDR complex components. (A–D) Streptavidin (SA) pulldown and co-immunopurification assays confirming interactions from Mass Spectrometric analyses. The BLRP tag is biotinylated in vivo allowing interaction with streptavidin.
    Figure Legend Snippet: Characterization of DDR complex components. (A–D) Streptavidin (SA) pulldown and co-immunopurification assays confirming interactions from Mass Spectrometric analyses. The BLRP tag is biotinylated in vivo allowing interaction with streptavidin.

    Techniques Used: Immu-Puri, In Vivo

    26) Product Images from "The Dual Use of RNA Aptamer Sequences for Affinity Purification and Localization Studies of RNAs and RNA-Protein Complexes"

    Article Title: The Dual Use of RNA Aptamer Sequences for Affinity Purification and Localization Studies of RNAs and RNA-Protein Complexes

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-61779-005-8_26

    Localization of yeast RNase P. The RNA subunit of the RNase P ribonucleoprotein (RPR1) is localized using a fluorophore labeled antisense oligo ( red ) targeting the streptavidin binding aptamer insertion into the RNA subunit (S1-RPR1). The U14 RNA is also visualized using an antisense oligo ( green ) and serves as a nucleolar marker. The overlap ( yellow ) shows that the wild type RNA is predominantly nucleolar. The complex becomes mislocalized to the nucleoplasm ( blue with permission of Cold Spring Harbor Press.
    Figure Legend Snippet: Localization of yeast RNase P. The RNA subunit of the RNase P ribonucleoprotein (RPR1) is localized using a fluorophore labeled antisense oligo ( red ) targeting the streptavidin binding aptamer insertion into the RNA subunit (S1-RPR1). The U14 RNA is also visualized using an antisense oligo ( green ) and serves as a nucleolar marker. The overlap ( yellow ) shows that the wild type RNA is predominantly nucleolar. The complex becomes mislocalized to the nucleoplasm ( blue with permission of Cold Spring Harbor Press.

    Techniques Used: Labeling, Binding Assay, Marker

    Insertion of the streptavidin binding aptamer into ribonucleoprotein complexes. ( Yeast RNase P and RNase MRP ) Aptamer insertions into the yeast RNase MRP and RNase P holoenzymes have been used to purify these ribonucleoprotein complexes. The yeast RNase P has been extensively tagged, initially with the full SELEX derived sequence (shown) and subsequently with the minimal streptavidin binding aptamer sequence. The yeast RNase P can be tagged in four different locations that are indicated (*). In each case, these positions are known to be phylogenetically variable and solvent exposed and the aptamer insertion does not alter the growth or pre-tRNA processing significantly. ( Yeast telomerase ) The yeast telomerase was tagged at two positions, the successful aptamer insertion is shown and the unsuccessful position is also indicated (#). The purified telomerase was subsequently shown to be active in a telomerase assay. ( Bacterial ribosomes ) The streptavidin aptamer was used to specifically isolate ribosomal subunits containing a known lethal mutation. Coexpression of the lethal mutation alongside the wildtype sequence was necessary to sustain bacterial growth.
    Figure Legend Snippet: Insertion of the streptavidin binding aptamer into ribonucleoprotein complexes. ( Yeast RNase P and RNase MRP ) Aptamer insertions into the yeast RNase MRP and RNase P holoenzymes have been used to purify these ribonucleoprotein complexes. The yeast RNase P has been extensively tagged, initially with the full SELEX derived sequence (shown) and subsequently with the minimal streptavidin binding aptamer sequence. The yeast RNase P can be tagged in four different locations that are indicated (*). In each case, these positions are known to be phylogenetically variable and solvent exposed and the aptamer insertion does not alter the growth or pre-tRNA processing significantly. ( Yeast telomerase ) The yeast telomerase was tagged at two positions, the successful aptamer insertion is shown and the unsuccessful position is also indicated (#). The purified telomerase was subsequently shown to be active in a telomerase assay. ( Bacterial ribosomes ) The streptavidin aptamer was used to specifically isolate ribosomal subunits containing a known lethal mutation. Coexpression of the lethal mutation alongside the wildtype sequence was necessary to sustain bacterial growth.

    Techniques Used: Binding Assay, Derivative Assay, Sequencing, Purification, Telomerase Assay, Mutagenesis

    The streptavidin binding RNA aptamer. ( a ) The original SELEX derived streptavidin binding sequence is shown along with the minimal streptavidin binding aptamer ( shaded ). The aptamer was derived from a population of species and the consensus sequence from multiple isolated clones is shown. ( b ) The streptavidin binding aptamer has been used for affinity purifications of ribonucleoprotein complexes ( left ) and the sequence has also been targeted as a unique hybridization region for fluorescent in situ hybridizations to localize ribonucleoprotein complexes ( right ).
    Figure Legend Snippet: The streptavidin binding RNA aptamer. ( a ) The original SELEX derived streptavidin binding sequence is shown along with the minimal streptavidin binding aptamer ( shaded ). The aptamer was derived from a population of species and the consensus sequence from multiple isolated clones is shown. ( b ) The streptavidin binding aptamer has been used for affinity purifications of ribonucleoprotein complexes ( left ) and the sequence has also been targeted as a unique hybridization region for fluorescent in situ hybridizations to localize ribonucleoprotein complexes ( right ).

    Techniques Used: Binding Assay, Derivative Assay, Sequencing, Isolation, Clone Assay, Hybridization, In Situ

    27) Product Images from "Berberine Targets AP-2/hTERT, NF-?B/COX-2, HIF-1?/VEGF and Cytochrome-c/Caspase Signaling to Suppress Human Cancer Cell Growth"

    Article Title: Berberine Targets AP-2/hTERT, NF-?B/COX-2, HIF-1?/VEGF and Cytochrome-c/Caspase Signaling to Suppress Human Cancer Cell Growth

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0069240

    BBR inhibited AP-2/hTERT signaling. ( A – C ) Human NSCLC A549 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the AP-2 and hTERT proteins ( A ) and mRNA ( B ) were analyzed by Western blotting and RT-PCR, respectively. GAPDH were used as controls for sample loading. The binding of AP-2 to hTERT promoter probe ( C ) was analyzed by a streptavidin-agarose pulldown assay. ( D ) A549 cells were transfected with an AP-2 siRNA or an AP-2-expressing vector for 24 hours, and then treated with BBR (100 µM). At 48 hours after treatment, protein expression and cell viability were determined by Western blot and MTT assay, respectively. The percent cell viability in each treatment group was calculated relative to cells treated with the vehicle control. The data are presented as the mean ± SD of three separate experiments. *, P
    Figure Legend Snippet: BBR inhibited AP-2/hTERT signaling. ( A – C ) Human NSCLC A549 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the AP-2 and hTERT proteins ( A ) and mRNA ( B ) were analyzed by Western blotting and RT-PCR, respectively. GAPDH were used as controls for sample loading. The binding of AP-2 to hTERT promoter probe ( C ) was analyzed by a streptavidin-agarose pulldown assay. ( D ) A549 cells were transfected with an AP-2 siRNA or an AP-2-expressing vector for 24 hours, and then treated with BBR (100 µM). At 48 hours after treatment, protein expression and cell viability were determined by Western blot and MTT assay, respectively. The percent cell viability in each treatment group was calculated relative to cells treated with the vehicle control. The data are presented as the mean ± SD of three separate experiments. *, P

    Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Transfection, Expressing, Plasmid Preparation, MTT Assay

    BBR inhibited NF-κB/COX-2 signaling. ( A ) Human A549 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the COX-2 protein was analyzed by Western blotting. GAPDH were used as controls for sample loading. ( B ) A549 cells were pretreated with the COX-2 selective inhibitor celecoxib (CB, 20 µM) for 24 hours, and then treated with BBR (20 µM). At 48 hours after treatment, cell viability was determined by MTT analysis. The percent cell viability in each treatment group was calculated relative to cells treated with the vehicle control. ( C ) A549 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the binding of p50 and p65 to COX-2 promoter probe was analyzed by a streptavidin-agarose pulldown assay. ( D ) A549 cells were treated with BBR (100 nM). At 48 hours after treatment, the effect of BBR on NF-κB p65 and p50 translocation was analyzed by immunofluorescence assay. The data are presented as the mean ± SD of three separate experiments. *, P
    Figure Legend Snippet: BBR inhibited NF-κB/COX-2 signaling. ( A ) Human A549 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the COX-2 protein was analyzed by Western blotting. GAPDH were used as controls for sample loading. ( B ) A549 cells were pretreated with the COX-2 selective inhibitor celecoxib (CB, 20 µM) for 24 hours, and then treated with BBR (20 µM). At 48 hours after treatment, cell viability was determined by MTT analysis. The percent cell viability in each treatment group was calculated relative to cells treated with the vehicle control. ( C ) A549 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the binding of p50 and p65 to COX-2 promoter probe was analyzed by a streptavidin-agarose pulldown assay. ( D ) A549 cells were treated with BBR (100 nM). At 48 hours after treatment, the effect of BBR on NF-κB p65 and p50 translocation was analyzed by immunofluorescence assay. The data are presented as the mean ± SD of three separate experiments. *, P

    Techniques Used: Western Blot, MTT Assay, Binding Assay, Translocation Assay, Immunofluorescence

    28) Product Images from "Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin"

    Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.118.113159

    MKK7 γ , but not MKK7 β , binds to CaN through a direct PxIxIT motif interaction. (A) Diagram showing the experimental design for detecting PVIVIT-GFP disruption of anti-myc co-IP of CaNA-myc and Flag-MKK7 γ and -MKK7 β in HEK-293 cells. Transfection of HEK-293 cells with higher amounts of plasmid DNA leading to higher levels of VIVIT-GFP expression [middle panel, immunoblotting (IB): GFP] are required to disrupt anti-myc co-IP of Flag-MKK7 γ 1 (B) versus Flag-MKK7 β 1 (C) (top panel, IB: Flag) with CaNA-myc (bottom panel; IB: myc). (D) Diagram showing the experimental design for detecting precipitation of CaNA-myc by biotinylated MKK7 β or MKK7 γ 35–52 peptides coupled to streptavidin agarose beads. (E) MKK7 γ 35–52 and PVIVIT biotinylated peptides precipitate CaNA-myc from HEK-293 cell extracts. MKK7 β 35–52 and negative control (Ht31) biotinylated peptides do not precipitate CaNA-myc. (F) The MKK7 γ 35–52 peptide competes with an affinity similar to PVIVIT for fluorescent-PVIVIT binding to CaN measured by fluorescence polarization (mP). MKK7 β 35–52 only competes for fluorescent PVIVIT binding to CaN at much higher concentrations. All data are representative of three independent experiments.
    Figure Legend Snippet: MKK7 γ , but not MKK7 β , binds to CaN through a direct PxIxIT motif interaction. (A) Diagram showing the experimental design for detecting PVIVIT-GFP disruption of anti-myc co-IP of CaNA-myc and Flag-MKK7 γ and -MKK7 β in HEK-293 cells. Transfection of HEK-293 cells with higher amounts of plasmid DNA leading to higher levels of VIVIT-GFP expression [middle panel, immunoblotting (IB): GFP] are required to disrupt anti-myc co-IP of Flag-MKK7 γ 1 (B) versus Flag-MKK7 β 1 (C) (top panel, IB: Flag) with CaNA-myc (bottom panel; IB: myc). (D) Diagram showing the experimental design for detecting precipitation of CaNA-myc by biotinylated MKK7 β or MKK7 γ 35–52 peptides coupled to streptavidin agarose beads. (E) MKK7 γ 35–52 and PVIVIT biotinylated peptides precipitate CaNA-myc from HEK-293 cell extracts. MKK7 β 35–52 and negative control (Ht31) biotinylated peptides do not precipitate CaNA-myc. (F) The MKK7 γ 35–52 peptide competes with an affinity similar to PVIVIT for fluorescent-PVIVIT binding to CaN measured by fluorescence polarization (mP). MKK7 β 35–52 only competes for fluorescent PVIVIT binding to CaN at much higher concentrations. All data are representative of three independent experiments.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Expressing, Negative Control, Binding Assay, Fluorescence

    29) Product Images from "Sequential and ?-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth"

    Article Title: Sequential and ?-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.060830

    BTC-ICD remains attached to the membrane compartment through palmitoylation. ( A ) IMPE-BTC-WT cells were lysed in lysis buffer with or without 50 mM NEM and cellular BTC isoforms precipitated with anti-HA agarose prior to acyl-biotin exchange labeling as described in the Materials and Methods. Eluted biotinylated proteins were re-precipitated with streptavidin-agarose and analyzed by western blot with anti-HA antibody (upper panel). Separate samples were directly analyzed in western blot with anti-HA antibody as loading controls (lower panel). ( B ) IMPE cells expressing either BTC-WT, BTC-Palm or BTC-ΔE were incubated in fatty-acid-free DMEM supplemented with 200 μCi of [ 3 H]palmitic acid for 6 hours at 37°C. Cell lysates were precipitated with anti-HA agarose and analyzed in western blot with anti-HA antibody (right panel) or membranes were used for autoradiography (left panel). ( C ) IMPE-BTC-WT cells were homogenized in the presence or absence of 1 M hydroxylamine-HCl (hydroxylamine) prior to pelleting membranes as described in the Materials and Methods. Cell lysates from membrane fractions and supernatants (S/N) were precipitated with anti-HA agarose and analyzed by western blotting with anti-HA antibody (upper panel: short exposure; lower panel: long exposure shows only BTC-CTF and BTC-ICD).
    Figure Legend Snippet: BTC-ICD remains attached to the membrane compartment through palmitoylation. ( A ) IMPE-BTC-WT cells were lysed in lysis buffer with or without 50 mM NEM and cellular BTC isoforms precipitated with anti-HA agarose prior to acyl-biotin exchange labeling as described in the Materials and Methods. Eluted biotinylated proteins were re-precipitated with streptavidin-agarose and analyzed by western blot with anti-HA antibody (upper panel). Separate samples were directly analyzed in western blot with anti-HA antibody as loading controls (lower panel). ( B ) IMPE cells expressing either BTC-WT, BTC-Palm or BTC-ΔE were incubated in fatty-acid-free DMEM supplemented with 200 μCi of [ 3 H]palmitic acid for 6 hours at 37°C. Cell lysates were precipitated with anti-HA agarose and analyzed in western blot with anti-HA antibody (right panel) or membranes were used for autoradiography (left panel). ( C ) IMPE-BTC-WT cells were homogenized in the presence or absence of 1 M hydroxylamine-HCl (hydroxylamine) prior to pelleting membranes as described in the Materials and Methods. Cell lysates from membrane fractions and supernatants (S/N) were precipitated with anti-HA agarose and analyzed by western blotting with anti-HA antibody (upper panel: short exposure; lower panel: long exposure shows only BTC-CTF and BTC-ICD).

    Techniques Used: Lysis, Labeling, Western Blot, Expressing, Incubation, Autoradiography

    30) Product Images from "VASP promotes TGF-β activation of hepatic stellate cells by regulating Rab11 dependent plasma membrane targeting of TGF-β receptors"

    Article Title: VASP promotes TGF-β activation of hepatic stellate cells by regulating Rab11 dependent plasma membrane targeting of TGF-β receptors

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.27251

    VASP knockdown inhibits TβRII recycling and TGF-β1 mediated downregulation of TβRII in HSCs (A) Serum starved HSCs were moved to 4°C and subjected to biotinylation of cell surface proteins using cleavable biotin that can be de-biotinylated by glutathione (50 mM) supplemented in cell culture medium. Cells cultured at 37°C for indicated times were harvested for streptavidin agarose pull down and WB for TβRII. Biotinylated TβRII detected in cells with glutathione treatment was internalized TβRII only and TβRII in cells without glutathione treatment was the sum of internalized and recycled (plasma membrane) TβRII. Densitometric analysis was done using the Image J software. VASP knockdown in HSCs reduced recycling of TβRII back to the plasma membrane at 60 min of incubation at 37°C (left, bottom row). It did not affect internalization of TβRII at 30 min (right, bottom row). AU, arbitrary unit. Data represent multiple repeats. (B) HSCs that were transfected with two different Rab11 siRNAs were subjected to biotinylation followed by streptavidin agarose pull down and WB for TβRII. Rab11 knockdown in HSCs significantly reduced cell surface TβRII. * p
    Figure Legend Snippet: VASP knockdown inhibits TβRII recycling and TGF-β1 mediated downregulation of TβRII in HSCs (A) Serum starved HSCs were moved to 4°C and subjected to biotinylation of cell surface proteins using cleavable biotin that can be de-biotinylated by glutathione (50 mM) supplemented in cell culture medium. Cells cultured at 37°C for indicated times were harvested for streptavidin agarose pull down and WB for TβRII. Biotinylated TβRII detected in cells with glutathione treatment was internalized TβRII only and TβRII in cells without glutathione treatment was the sum of internalized and recycled (plasma membrane) TβRII. Densitometric analysis was done using the Image J software. VASP knockdown in HSCs reduced recycling of TβRII back to the plasma membrane at 60 min of incubation at 37°C (left, bottom row). It did not affect internalization of TβRII at 30 min (right, bottom row). AU, arbitrary unit. Data represent multiple repeats. (B) HSCs that were transfected with two different Rab11 siRNAs were subjected to biotinylation followed by streptavidin agarose pull down and WB for TβRII. Rab11 knockdown in HSCs significantly reduced cell surface TβRII. * p

    Techniques Used: Cell Culture, Western Blot, Software, Incubation, Transfection

    31) Product Images from "Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells"

    Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

    Journal: The Biochemical journal

    doi: 10.1042/BCJ20160203

    Cathepsin B inhibitor increases the amount of FCF-induced ubiquitylated forms of ErbB2 in cell lysates but not in the plasma membrane fraction HGE-20 cells were incubated with the indicated inhibitors for 6 hours, followed by basolateral biotinylation of surface proteins, streptavidin extraction, and western blot using anti-ErbB2 antibodies. Na + ,K + -ATPase β 1 subunit was used as a loading control. ErbB2 protein levels in the membrane fraction were not protected by any of the indicated inhibitors in the presence of FCF (A). In total cell lysates, ubiquitylated ErbB2, seen as an increased density running above the main ErbB2 band (arrowhead), was increased in the presence of cathepsin B inhibitor CA074-me (B). ErbB2 was immunoprecipitated, followed by western blot using anti-ubiquitin, then anti-ErbB2 antibodies, confirming a significant increase in ubiquitylated forms of ErbB2 in the presence of CA074-me and also showing a moderate increase in the presence of lactacystin (C). Quantification for each blot is shown to the right. Error bars, s.d., n =4 independent experiments, statistics done by Student’s t-test, ns-not significant, * - significant difference from the no-FCF control, P
    Figure Legend Snippet: Cathepsin B inhibitor increases the amount of FCF-induced ubiquitylated forms of ErbB2 in cell lysates but not in the plasma membrane fraction HGE-20 cells were incubated with the indicated inhibitors for 6 hours, followed by basolateral biotinylation of surface proteins, streptavidin extraction, and western blot using anti-ErbB2 antibodies. Na + ,K + -ATPase β 1 subunit was used as a loading control. ErbB2 protein levels in the membrane fraction were not protected by any of the indicated inhibitors in the presence of FCF (A). In total cell lysates, ubiquitylated ErbB2, seen as an increased density running above the main ErbB2 band (arrowhead), was increased in the presence of cathepsin B inhibitor CA074-me (B). ErbB2 was immunoprecipitated, followed by western blot using anti-ubiquitin, then anti-ErbB2 antibodies, confirming a significant increase in ubiquitylated forms of ErbB2 in the presence of CA074-me and also showing a moderate increase in the presence of lactacystin (C). Quantification for each blot is shown to the right. Error bars, s.d., n =4 independent experiments, statistics done by Student’s t-test, ns-not significant, * - significant difference from the no-FCF control, P

    Techniques Used: Incubation, Western Blot, Immunoprecipitation

    FCF induces ubiquitylation of ErbB2 at the plasma membrane There was no difference in FCF-induced decrease in ErbB2 in the presence or absence of cycloheximide, as shown by western blot of total HGE-20 cell lysate, suggesting the effect of FCF is on mature ErbB2 protein (A). Incubation with FCF or vehicle was completed for 12 hours with or without cycloheximide, followed by immunoprecipitation with ErbB2 antibodies. Western blot using ubiquitin antibodies, followed ErbB2 antibodies, showed increased ubiquitylation of ErbB2 in the presence of FCF, independent of new protein synthesis (B). Ubiquitylation induced by FCF is specific to ErbB2, as shown by western blot comparison of ErbB2 immunoprecipitate and total cell lysate using ubiquitin antibodies; β-actin was used as a loading control (C). Cells were incubated with FCF or vehicle, surface proteins were biotinylated from basolateral side, followed by immunoprecipitation with ErbB2 antibodies (IP1), then boiling of eluted proteins in SDS to remove interacting proteins, followed by dilution in non-ionic detergent and repeat immunoprecipitation with ErbB2 antibodies (IP2). Loss of septin-9 and cofilin signals on western blot in IP2 confirms dissociation of ErbB2 from interacting proteins. Increased ubiquitylation of ErbB2 is still seen in the absence of interacting proteins, demonstrating that ErbB2 itself is ubiquitylated. Proteins eluted from the second IP by boiling in SDS were diluted in non-ionic detergent and biotinylated ErbB2 was isolated by streptavidin extraction (SA). As shown by western blot with ubiquitin antibodies, the increased ubiquitylation of ErbB2 induced by FCF is seen in this fraction, demonstrating ubiquitylation occurs at the plasma membrane. The graph shows the FCF-increased ubiquitylation seen in each experiment as compared to control (D). Ub-ubiquitylated ErbB2, UM-unmodified ErbB2, IgG-immunoprecipitation control without cell lysate, SA-streptavidin.
    Figure Legend Snippet: FCF induces ubiquitylation of ErbB2 at the plasma membrane There was no difference in FCF-induced decrease in ErbB2 in the presence or absence of cycloheximide, as shown by western blot of total HGE-20 cell lysate, suggesting the effect of FCF is on mature ErbB2 protein (A). Incubation with FCF or vehicle was completed for 12 hours with or without cycloheximide, followed by immunoprecipitation with ErbB2 antibodies. Western blot using ubiquitin antibodies, followed ErbB2 antibodies, showed increased ubiquitylation of ErbB2 in the presence of FCF, independent of new protein synthesis (B). Ubiquitylation induced by FCF is specific to ErbB2, as shown by western blot comparison of ErbB2 immunoprecipitate and total cell lysate using ubiquitin antibodies; β-actin was used as a loading control (C). Cells were incubated with FCF or vehicle, surface proteins were biotinylated from basolateral side, followed by immunoprecipitation with ErbB2 antibodies (IP1), then boiling of eluted proteins in SDS to remove interacting proteins, followed by dilution in non-ionic detergent and repeat immunoprecipitation with ErbB2 antibodies (IP2). Loss of septin-9 and cofilin signals on western blot in IP2 confirms dissociation of ErbB2 from interacting proteins. Increased ubiquitylation of ErbB2 is still seen in the absence of interacting proteins, demonstrating that ErbB2 itself is ubiquitylated. Proteins eluted from the second IP by boiling in SDS were diluted in non-ionic detergent and biotinylated ErbB2 was isolated by streptavidin extraction (SA). As shown by western blot with ubiquitin antibodies, the increased ubiquitylation of ErbB2 induced by FCF is seen in this fraction, demonstrating ubiquitylation occurs at the plasma membrane. The graph shows the FCF-increased ubiquitylation seen in each experiment as compared to control (D). Ub-ubiquitylated ErbB2, UM-unmodified ErbB2, IgG-immunoprecipitation control without cell lysate, SA-streptavidin.

    Techniques Used: Western Blot, Incubation, Immunoprecipitation, Isolation

    32) Product Images from "Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules"

    Article Title: Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules

    Journal: The EMBO Journal

    doi: 10.1093/emboj/21.7.1650

    Fig. 4. The CD1d–class II association exists in MIICs and on the cell surface. ( A ) C1R.CD1d cells were disrupted by a ball-bearing homogenizer and fractionated using a Percoll density gradient. Aliquots of each fraction were analyzed by SDS–PAGE followed by immunoblotting with D5, R.DRAB (rabbit anti-DR), DM323 (rabbit anti-DM) and MaP.ERp57 (anti-ERp57 mAb). Fraction 1 corresponds to the top of the gradient. ( B ) CD1d–class II complexes were immunoprecipitated from Brij 98-solubilized fractions using L243 and analyzed by SDS–PAGE followed by immunoblotting with D5 or R.DRAB. ( C ) C1R.CD1d cells were radiolabeled for 15 min and chased for 30 min (lanes 1 and 3) or 5 h (lanes 2 and 4). Cell surface proteins were biotinylated with sulfo-NHS-SS-biotin (see Materials and methods) prior to extraction in 1% Brij 98. The extracts were immunoprecipitated with either 51.1.3 (lanes 1 and 2) or L243 (lanes 3 and 4). Precipitated proteins were dissociated from the beads in non-reducing elution buffer containing 1% SDS and the eluates re-precipitated with streptavidin–agarose prior to separation by 12% SDS–PAGE. The bands corresponding to CD1d, and DRα and β chains, are marked on the right of the gel.
    Figure Legend Snippet: Fig. 4. The CD1d–class II association exists in MIICs and on the cell surface. ( A ) C1R.CD1d cells were disrupted by a ball-bearing homogenizer and fractionated using a Percoll density gradient. Aliquots of each fraction were analyzed by SDS–PAGE followed by immunoblotting with D5, R.DRAB (rabbit anti-DR), DM323 (rabbit anti-DM) and MaP.ERp57 (anti-ERp57 mAb). Fraction 1 corresponds to the top of the gradient. ( B ) CD1d–class II complexes were immunoprecipitated from Brij 98-solubilized fractions using L243 and analyzed by SDS–PAGE followed by immunoblotting with D5 or R.DRAB. ( C ) C1R.CD1d cells were radiolabeled for 15 min and chased for 30 min (lanes 1 and 3) or 5 h (lanes 2 and 4). Cell surface proteins were biotinylated with sulfo-NHS-SS-biotin (see Materials and methods) prior to extraction in 1% Brij 98. The extracts were immunoprecipitated with either 51.1.3 (lanes 1 and 2) or L243 (lanes 3 and 4). Precipitated proteins were dissociated from the beads in non-reducing elution buffer containing 1% SDS and the eluates re-precipitated with streptavidin–agarose prior to separation by 12% SDS–PAGE. The bands corresponding to CD1d, and DRα and β chains, are marked on the right of the gel.

    Techniques Used: SDS Page, Immunoprecipitation

    Fig. 2. Human CD1d associates with MHC class II molecules. ( A ) C1R.CD1d cells were labeled with [ 35 S]methionine for 5 h, extracted in 1% Brij 98 and immunoprecipitated with a control mAb W6/32 (anti-class I, lanes 1 and 3), 51.1.3 (anti-CD1d, lane 2), L243 (anti-HLA DRαβ dimer, lane 4) or DA6.147 (anti-DRα-chain, lane 5). SDS/DTT-eluted material was re-precipitated with DA6.147 (lanes 1 and 2) or D5 (anti-CD1d heavy chain, lanes 3–5), and analyzed by 12% SDS–PAGE. ( B ) The interaction of CD1d with class II molecules exists in cells before lysis. In lanes 1 and 2, C1R cells (2 × 10 6 cells/lane), metabolically labeled with [ 35 S]methionine for 5 h, were mixed with unlabeled C1R.CD1d cells (2 × 10 6 cells/lane) and lysed in 1% Brij 98. The lysates were immunoprecipitated with a control mAb GAP.A3 or mAb 51.1.3. In lanes 3 and 4, [ 35 S]methionine-labeled C1R.CD1d cells (2 × 10 6 cells/lane) were lysed, and immunoprecipitated as in lanes 1 and 2. The immunoprecipitates were analyzed by 12% SDS–PAGE. The bands corresponding to CD1d heavy chain and class II αβ chains are indicated. ( C ) .221.TMCD1d.f cells were lysed in 2% Brij 98 and the lysate was passed through an M2-conjugated anti-FLAG affinity column. CD1d and class II complexes were eluted with FLAG peptides. Aliquots of the eluate (top panels) and flow-through (middle and bottom panels) were used for immunoprecipitation with GAP.A3, MaP.CD82 (anti-CD82), H5C6 (anti-CD63), JS-81 (anti-CD81) or L243. HLA-DRα chain was detected by western blotting with R.DRAB (anti-HLA-DRαβ). The bands corresponding to DRα are indicated on the left. ( D ) Monocyte-derived DCs (top panels) and monocyte-depleted lymphocytes (middle panels) were lysed in 2% Brij 98, and the extracts were incubated sequentially with beads conjugated with 28-8-6S (C1), a negative control mAb and with L243. The DR-associated CD1d was eluted with 1% C 12 E 9 , ethanol precipitated, separated by SDS–PAGE and detected by western blotting with biotinylated D5 and HRP-conjugated streptavidin (lanes 1 and 2). To identify free CD1d, the supernatants from the initial L243 immunoprecipitations (the total for DCs, 1/20 of the total for monocyte-depleted lymphocytes) were re-immunoprecipitated with either a negative control mAb, 28-14-8S (C2) or 51.1.3 and detected as above (lanes 3 and 4). As a positive control, C1R.CD1d cells (bottom panels) were subjected to the same procedure. The bands corresponding to CD1d heavy chain are indicated on the left.
    Figure Legend Snippet: Fig. 2. Human CD1d associates with MHC class II molecules. ( A ) C1R.CD1d cells were labeled with [ 35 S]methionine for 5 h, extracted in 1% Brij 98 and immunoprecipitated with a control mAb W6/32 (anti-class I, lanes 1 and 3), 51.1.3 (anti-CD1d, lane 2), L243 (anti-HLA DRαβ dimer, lane 4) or DA6.147 (anti-DRα-chain, lane 5). SDS/DTT-eluted material was re-precipitated with DA6.147 (lanes 1 and 2) or D5 (anti-CD1d heavy chain, lanes 3–5), and analyzed by 12% SDS–PAGE. ( B ) The interaction of CD1d with class II molecules exists in cells before lysis. In lanes 1 and 2, C1R cells (2 × 10 6 cells/lane), metabolically labeled with [ 35 S]methionine for 5 h, were mixed with unlabeled C1R.CD1d cells (2 × 10 6 cells/lane) and lysed in 1% Brij 98. The lysates were immunoprecipitated with a control mAb GAP.A3 or mAb 51.1.3. In lanes 3 and 4, [ 35 S]methionine-labeled C1R.CD1d cells (2 × 10 6 cells/lane) were lysed, and immunoprecipitated as in lanes 1 and 2. The immunoprecipitates were analyzed by 12% SDS–PAGE. The bands corresponding to CD1d heavy chain and class II αβ chains are indicated. ( C ) .221.TMCD1d.f cells were lysed in 2% Brij 98 and the lysate was passed through an M2-conjugated anti-FLAG affinity column. CD1d and class II complexes were eluted with FLAG peptides. Aliquots of the eluate (top panels) and flow-through (middle and bottom panels) were used for immunoprecipitation with GAP.A3, MaP.CD82 (anti-CD82), H5C6 (anti-CD63), JS-81 (anti-CD81) or L243. HLA-DRα chain was detected by western blotting with R.DRAB (anti-HLA-DRαβ). The bands corresponding to DRα are indicated on the left. ( D ) Monocyte-derived DCs (top panels) and monocyte-depleted lymphocytes (middle panels) were lysed in 2% Brij 98, and the extracts were incubated sequentially with beads conjugated with 28-8-6S (C1), a negative control mAb and with L243. The DR-associated CD1d was eluted with 1% C 12 E 9 , ethanol precipitated, separated by SDS–PAGE and detected by western blotting with biotinylated D5 and HRP-conjugated streptavidin (lanes 1 and 2). To identify free CD1d, the supernatants from the initial L243 immunoprecipitations (the total for DCs, 1/20 of the total for monocyte-depleted lymphocytes) were re-immunoprecipitated with either a negative control mAb, 28-14-8S (C2) or 51.1.3 and detected as above (lanes 3 and 4). As a positive control, C1R.CD1d cells (bottom panels) were subjected to the same procedure. The bands corresponding to CD1d heavy chain are indicated on the left.

    Techniques Used: Labeling, Immunoprecipitation, SDS Page, Lysis, Metabolic Labelling, Affinity Column, Flow Cytometry, Western Blot, Derivative Assay, Incubation, Negative Control, Positive Control

    33) Product Images from "Analysis of metabotropic glutamate receptor 7 as a potential substrate for SUMOylation"

    Article Title: Analysis of metabotropic glutamate receptor 7 as a potential substrate for SUMOylation

    Journal: Neuroscience Letters

    doi: 10.1016/j.neulet.2011.01.032

    Expression and surface trafficking of a wild-type and non-SUMOylatable SEP-mGluR7 in HEK293 cells: (A) SEP-mGluR7 was transfected into HEK293 cells. Thirty-six hours post-transfection, surface proteins were labelled with biotin, the cells lysed and biotinylated surface proteins were isolated on streptavidin beads and subjected to Western blotting alongside total lysate for GFP (SEP). (B) SEP-mGluR7 was transfected into 13DIV primary cortical neurons and imaged for SEP expression 5 days later (upper panel). SEP fluorescence was reduced with a transient exposure to pH 6.0 buffer consistent with the majority of the signal arising from surface expressed SEP-mGluR7. Application of ammonium chloride, which collapses pH gradients across the plasma membrane and transiently equilibrates intracellular compartments to the extracellular pH increased fluorescence showing the total SEP signal (pH 7.4) or minimum signal (pH 6). The fluorescence of the region boxed in the upper panel is shown graphically in the lower panel. (C) SEP-mGluR7 is functional in HEK293 cells, as assayed for its ability to activate the ERK pathway. Data are the mean ± SEM, ** p
    Figure Legend Snippet: Expression and surface trafficking of a wild-type and non-SUMOylatable SEP-mGluR7 in HEK293 cells: (A) SEP-mGluR7 was transfected into HEK293 cells. Thirty-six hours post-transfection, surface proteins were labelled with biotin, the cells lysed and biotinylated surface proteins were isolated on streptavidin beads and subjected to Western blotting alongside total lysate for GFP (SEP). (B) SEP-mGluR7 was transfected into 13DIV primary cortical neurons and imaged for SEP expression 5 days later (upper panel). SEP fluorescence was reduced with a transient exposure to pH 6.0 buffer consistent with the majority of the signal arising from surface expressed SEP-mGluR7. Application of ammonium chloride, which collapses pH gradients across the plasma membrane and transiently equilibrates intracellular compartments to the extracellular pH increased fluorescence showing the total SEP signal (pH 7.4) or minimum signal (pH 6). The fluorescence of the region boxed in the upper panel is shown graphically in the lower panel. (C) SEP-mGluR7 is functional in HEK293 cells, as assayed for its ability to activate the ERK pathway. Data are the mean ± SEM, ** p

    Techniques Used: Expressing, Transfection, Isolation, Western Blot, Fluorescence, Functional Assay

    34) Product Images from "Characterization of the Ubiquinone Binding Site in Alternative NADH-Quinone Oxidoreductase of Saccharomyces cerevisiae by Photoaffinity Labeling †"

    Article Title: Characterization of the Ubiquinone Binding Site in Alternative NADH-Quinone Oxidoreductase of Saccharomyces cerevisiae by Photoaffinity Labeling †

    Journal: Biochemistry

    doi: 10.1021/bi100005j

    Photoaffinity labeling of Ndi1 by the biotinylated azido-Qs ( 1 - 3 ). The UQ-free Ndi1 was photoirradiated with each azido-Q at an equivalent molar ratio (6.6 μg of protein/mL), and analyzed on 10% Laemmli’s gel with CBB stain and streptavidin-AP (0.5 μg of protein/lane for CBB stain and 0.1 μg of protein/lane for Western blotting). Data shown are representative of four independent experiments.
    Figure Legend Snippet: Photoaffinity labeling of Ndi1 by the biotinylated azido-Qs ( 1 - 3 ). The UQ-free Ndi1 was photoirradiated with each azido-Q at an equivalent molar ratio (6.6 μg of protein/mL), and analyzed on 10% Laemmli’s gel with CBB stain and streptavidin-AP (0.5 μg of protein/lane for CBB stain and 0.1 μg of protein/lane for Western blotting). Data shown are representative of four independent experiments.

    Techniques Used: Labeling, Staining, Western Blot

    35) Product Images from "Identification of a Heme Activation Site on the MD-2/TLR4 Complex"

    Article Title: Identification of a Heme Activation Site on the MD-2/TLR4 Complex

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.01370

    Heme binds poorly to W23A and W23A-S33A-Y34A mutant MD-2. WT MD-2, W23A MD-2, and W23A/S33A/Y34A MD-2 with Flag tags were expressed in CHO cells and the MD-2-containing media were used in heme-agarose and biotin-heme/streptavidin-agarose pull-down assays. Three days after transfection, the conditioned CHO media contained similar amounts of WT, W23A, and W23A/S33A/Y34A MD-2 on Western blots using Flag detection ( A , lanes 5, 6, and 7 and B , lanes 6, 7, and 8). Heme-agarose pulled down only 47% of the W23A and 36% of the W23A/S33A/Y34A MD-2 mutants compared to WT MD-2 ( A , lanes 2, 3, and 4 and C ). WT MD-2 incubated with control agarose without heme was not pulled down ( A , lane 1). Similarly, the biotin-heme/streptavidin-agarose pulled down only 32% of the W23A and 20% of the W23A/S33A/Y34A MD-2 mutants compared to WT MD-2 ( B , lanes 2, 3, and 4 and D ). WT MD-2 incubated with biotin-heme was not pulled down by agarose without streptavidin ( B , lane 1). As previously shown, the addition of excess unlabeled heme markedly reduced the amount of WT MD-2 pulled down by biotin-heme/streptavidin-agarose ( B , lane 5). The results shown in (A,B) are representative of four independent experiments. (C,D) show the results of quantitation of the Western blots. Bars are means + SD. * P
    Figure Legend Snippet: Heme binds poorly to W23A and W23A-S33A-Y34A mutant MD-2. WT MD-2, W23A MD-2, and W23A/S33A/Y34A MD-2 with Flag tags were expressed in CHO cells and the MD-2-containing media were used in heme-agarose and biotin-heme/streptavidin-agarose pull-down assays. Three days after transfection, the conditioned CHO media contained similar amounts of WT, W23A, and W23A/S33A/Y34A MD-2 on Western blots using Flag detection ( A , lanes 5, 6, and 7 and B , lanes 6, 7, and 8). Heme-agarose pulled down only 47% of the W23A and 36% of the W23A/S33A/Y34A MD-2 mutants compared to WT MD-2 ( A , lanes 2, 3, and 4 and C ). WT MD-2 incubated with control agarose without heme was not pulled down ( A , lane 1). Similarly, the biotin-heme/streptavidin-agarose pulled down only 32% of the W23A and 20% of the W23A/S33A/Y34A MD-2 mutants compared to WT MD-2 ( B , lanes 2, 3, and 4 and D ). WT MD-2 incubated with biotin-heme was not pulled down by agarose without streptavidin ( B , lane 1). As previously shown, the addition of excess unlabeled heme markedly reduced the amount of WT MD-2 pulled down by biotin-heme/streptavidin-agarose ( B , lane 5). The results shown in (A,B) are representative of four independent experiments. (C,D) show the results of quantitation of the Western blots. Bars are means + SD. * P

    Techniques Used: Mutagenesis, Transfection, Western Blot, Incubation, Quantitation Assay

    Heme binds to MD-2 in pull-down assays. Recombinant MD-2 and hemopexin (Hpx) were expressed by transfecting Chinese hamster ovary (CHO) cells with plasmids encoding human Flag-MD-2 or Flag-Hpx. Flag-Hpx served as a positive control for heme binding. After transfection, cells were washed and incubated for 72 h in protein-free CHO medium to allow the recombinant proteins to be transcribed, translated, and secreted into the CHO media. After 72 h, Flag-MD-2 ( A , lane 3 and C , lane 4) and Flag-Hpx ( B , lane 6 and D , lane 8) were present in the conditioned media of transfected CHO cells as demonstrated by a Western blot of the concentrated media with an anti-Flag primary antibody. (A,B) Heme-agarose pull-down assays. Conditioned CHO media containing (A) Flag-MD-2 or (B) Flag-Hpx were incubated overnight at 4°C with heme-agarose (lanes 2 and 5) or control agarose beads (lanes 1 and 4) and then pelleted, washed, and run on a Western blot with anti-Flag detection. (C,D) Streptavidin-agarose pull-down assays. Conditioned CHO media containing (C) Flag-MD-2 or (D) Flag-Hpx were incubated with 15 μM biotin-heme overnight at 4°C in the dark, then streptavidin-agarose (lanes 2, 3, 6, and 7) or control agarose (lanes 1 and 5) was added to the mixture for an additional 2 h at 4°C. After incubation, the agarose pellets were washed and run on Western blots with anti-Flag detection. To test the binding specificity of biotin-heme and recombinant Flag-MD-2 ( C , lane 3) and Flag-Hpx ( D , lane 7), conditioned CHO media were pre-incubated with an excess of unlabeled free heme (100 μM) for 2 h at 4°C before the incubation with 15 μM biotin-heme. The results shown are representative of four ( A and B ) and two ( C and D ) independent experiments, respectively.
    Figure Legend Snippet: Heme binds to MD-2 in pull-down assays. Recombinant MD-2 and hemopexin (Hpx) were expressed by transfecting Chinese hamster ovary (CHO) cells with plasmids encoding human Flag-MD-2 or Flag-Hpx. Flag-Hpx served as a positive control for heme binding. After transfection, cells were washed and incubated for 72 h in protein-free CHO medium to allow the recombinant proteins to be transcribed, translated, and secreted into the CHO media. After 72 h, Flag-MD-2 ( A , lane 3 and C , lane 4) and Flag-Hpx ( B , lane 6 and D , lane 8) were present in the conditioned media of transfected CHO cells as demonstrated by a Western blot of the concentrated media with an anti-Flag primary antibody. (A,B) Heme-agarose pull-down assays. Conditioned CHO media containing (A) Flag-MD-2 or (B) Flag-Hpx were incubated overnight at 4°C with heme-agarose (lanes 2 and 5) or control agarose beads (lanes 1 and 4) and then pelleted, washed, and run on a Western blot with anti-Flag detection. (C,D) Streptavidin-agarose pull-down assays. Conditioned CHO media containing (C) Flag-MD-2 or (D) Flag-Hpx were incubated with 15 μM biotin-heme overnight at 4°C in the dark, then streptavidin-agarose (lanes 2, 3, 6, and 7) or control agarose (lanes 1 and 5) was added to the mixture for an additional 2 h at 4°C. After incubation, the agarose pellets were washed and run on Western blots with anti-Flag detection. To test the binding specificity of biotin-heme and recombinant Flag-MD-2 ( C , lane 3) and Flag-Hpx ( D , lane 7), conditioned CHO media were pre-incubated with an excess of unlabeled free heme (100 μM) for 2 h at 4°C before the incubation with 15 μM biotin-heme. The results shown are representative of four ( A and B ) and two ( C and D ) independent experiments, respectively.

    Techniques Used: Recombinant, Positive Control, Binding Assay, Transfection, Incubation, Western Blot

    36) Product Images from "Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin"

    Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.118.113159

    MKK7 γ , but not MKK7 β , binds to CaN through a direct PxIxIT motif interaction. (A) Diagram showing the experimental design for detecting PVIVIT-GFP disruption of anti-myc co-IP of CaNA-myc and Flag-MKK7 γ and -MKK7 β in HEK-293 cells. Transfection of HEK-293 cells with higher amounts of plasmid DNA leading to higher levels of VIVIT-GFP expression [middle panel, immunoblotting (IB): GFP] are required to disrupt anti-myc co-IP of Flag-MKK7 γ 1 (B) versus Flag-MKK7 β 1 (C) (top panel, IB: Flag) with CaNA-myc (bottom panel; IB: myc). (D) Diagram showing the experimental design for detecting precipitation of CaNA-myc by biotinylated MKK7 β or MKK7 γ 35–52 peptides coupled to streptavidin agarose beads. (E) MKK7 γ 35–52 and PVIVIT biotinylated peptides precipitate CaNA-myc from HEK-293 cell extracts. MKK7 β 35–52 and negative control (Ht31) biotinylated peptides do not precipitate CaNA-myc. (F) The MKK7 γ 35–52 peptide competes with an affinity similar to PVIVIT for fluorescent-PVIVIT binding to CaN measured by fluorescence polarization (mP). MKK7 β 35–52 only competes for fluorescent PVIVIT binding to CaN at much higher concentrations. All data are representative of three independent experiments.
    Figure Legend Snippet: MKK7 γ , but not MKK7 β , binds to CaN through a direct PxIxIT motif interaction. (A) Diagram showing the experimental design for detecting PVIVIT-GFP disruption of anti-myc co-IP of CaNA-myc and Flag-MKK7 γ and -MKK7 β in HEK-293 cells. Transfection of HEK-293 cells with higher amounts of plasmid DNA leading to higher levels of VIVIT-GFP expression [middle panel, immunoblotting (IB): GFP] are required to disrupt anti-myc co-IP of Flag-MKK7 γ 1 (B) versus Flag-MKK7 β 1 (C) (top panel, IB: Flag) with CaNA-myc (bottom panel; IB: myc). (D) Diagram showing the experimental design for detecting precipitation of CaNA-myc by biotinylated MKK7 β or MKK7 γ 35–52 peptides coupled to streptavidin agarose beads. (E) MKK7 γ 35–52 and PVIVIT biotinylated peptides precipitate CaNA-myc from HEK-293 cell extracts. MKK7 β 35–52 and negative control (Ht31) biotinylated peptides do not precipitate CaNA-myc. (F) The MKK7 γ 35–52 peptide competes with an affinity similar to PVIVIT for fluorescent-PVIVIT binding to CaN measured by fluorescence polarization (mP). MKK7 β 35–52 only competes for fluorescent PVIVIT binding to CaN at much higher concentrations. All data are representative of three independent experiments.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Expressing, Negative Control, Binding Assay, Fluorescence

    37) Product Images from "Target-Based Screen Against a Periplasmic Serine Protease That Regulates Intrabacterial pH Homeostasis in Mycobacteriumtuberculosis"

    Article Title: Target-Based Screen Against a Periplasmic Serine Protease That Regulates Intrabacterial pH Homeostasis in Mycobacteriumtuberculosis

    Journal: ACS Chemical Biology

    doi: 10.1021/cb500746z

    BO43T probe to identify additional targets of BO43. (a) pH IB activity of BO43, BO43T, and 2DT on BCG at pH 4.5. (b) CFU activity of BO43, BO43T, and 2DT on BCG at pH 4.5. (c) MarP immunoblot (left) and streptavidin-fluorescence (right) of BO43T treated BCG following in situ labeling and pull-down; ★ indicates MarP homologue (Mb3695c), ★★ indicates HtrA1 homologue (Mb1255). Means ± S. E. M. of triplicate samples shown in a and b represent three independent experiments. Some error bars are smaller than the symbols.
    Figure Legend Snippet: BO43T probe to identify additional targets of BO43. (a) pH IB activity of BO43, BO43T, and 2DT on BCG at pH 4.5. (b) CFU activity of BO43, BO43T, and 2DT on BCG at pH 4.5. (c) MarP immunoblot (left) and streptavidin-fluorescence (right) of BO43T treated BCG following in situ labeling and pull-down; ★ indicates MarP homologue (Mb3695c), ★★ indicates HtrA1 homologue (Mb1255). Means ± S. E. M. of triplicate samples shown in a and b represent three independent experiments. Some error bars are smaller than the symbols.

    Techniques Used: Activity Assay, Fluorescence, In Situ, Labeling

    38) Product Images from "A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1)"

    Article Title: A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1)

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-11-0689

    Biochemical characterization of the tether complex. (A) Chromatographic separation of the axonemal salt extracts from FAP44C strain using an UnoQ anion-exchange column. The dynein species of each peak was identified according to the previous study ( Furuta et al. , 2009 ). FAP44 and FAP43 were detected using immunoblotting. Comigration of IDA f with FAP44 and FAP43 was clearly observed. (B) Immunoprecipitation of the FAP44 protein. Fractions containing purified IDA f , FAP44, and FAP43 were collected and BCCP-tagged FAP44 proteins were immunoprecipitated (IP) using streptavidin-agarose. As a negative control (biotin block), the streptavidin-agarose was blocked with 1 mM biocytin (biotin-lysine). IDA f was detected using the IC140 antibody. IDA f and FAP43 were co-immunoprecipitated with FAP44. (C) Sarkosyl fractionation of axonemal proteins. Axonemal proteins were extracted with various concentration of sarkosyl. Supernatant (sup) and precipitate (ppt) after centrifugation were analyzed by immunoblotting. Although the phenotypes of fap43 and fap244 are virtually wild type, extraction patterns showed destabilization of the tether complex.
    Figure Legend Snippet: Biochemical characterization of the tether complex. (A) Chromatographic separation of the axonemal salt extracts from FAP44C strain using an UnoQ anion-exchange column. The dynein species of each peak was identified according to the previous study ( Furuta et al. , 2009 ). FAP44 and FAP43 were detected using immunoblotting. Comigration of IDA f with FAP44 and FAP43 was clearly observed. (B) Immunoprecipitation of the FAP44 protein. Fractions containing purified IDA f , FAP44, and FAP43 were collected and BCCP-tagged FAP44 proteins were immunoprecipitated (IP) using streptavidin-agarose. As a negative control (biotin block), the streptavidin-agarose was blocked with 1 mM biocytin (biotin-lysine). IDA f was detected using the IC140 antibody. IDA f and FAP43 were co-immunoprecipitated with FAP44. (C) Sarkosyl fractionation of axonemal proteins. Axonemal proteins were extracted with various concentration of sarkosyl. Supernatant (sup) and precipitate (ppt) after centrifugation were analyzed by immunoblotting. Although the phenotypes of fap43 and fap244 are virtually wild type, extraction patterns showed destabilization of the tether complex.

    Techniques Used: Immunoprecipitation, Purification, Negative Control, Blocking Assay, Fractionation, Concentration Assay, Centrifugation

    39) Product Images from "Efficient T cell–B cell collaboration guides autoantibody epitope bias and onset of celiac disease"

    Article Title: Efficient T cell–B cell collaboration guides autoantibody epitope bias and onset of celiac disease

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

    doi: 10.1073/pnas.1901561116

    Binding characteristics of mAbs targeting non–N-terminal TG2 domains. ( A ). ( B ) Surface plasmon resonance sensorgrams showing change in response units (RU) caused by binding and dissociation of Fab fragments using different conformational states of TG2. When using TG2 covalently linked to an active-site inhibitor (iTG2) in the presence of Ca 2+ , only antibodies recognizing core-domain epitopes bound detectably, whereas all but one antibody showed binding in the presence of GDP. This antibody could still bind TG2 in the absence of effector molecules. ( C ) Comparison of TG2 affinities between Fab fragments specific to N-terminal ( n = 9) and non–N-terminal ( n = 7) epitopes. Plotted values were obtained with the TG2 conformation that, in each case, gave the highest affinity. Column heights indicate mean affinity, and statistical difference was evaluated using an unpaired t test. ( D ) Immunofluorescence detection of mAbs bound to human recombinant TG3/TG2, which was immobilized on mouse TG2 KO small intestinal tissue sections. ( E ) Representative flow cytometry plot ( Left ) and mean distribution of TG2-reactive plasma cells (PCs) between populations targeting C2 and non-C2 epitopes in three celiac patients ( Right ). The two populations were identified by costaining with full-length TG2 and a truncated TG2 variant consisting of the C2 domain only. ( F ) Western blots showing cross-link formation between TG2 and a biotinylated 33mer gluten peptide resulting in high-molecular-weight bands. Cross-linked TG2-gluten complexes were immunoprecipitated (IP) with the indicated mAbs, and protein bands were visualized using either anti-TG2 mAb or streptavidin (SA).
    Figure Legend Snippet: Binding characteristics of mAbs targeting non–N-terminal TG2 domains. ( A ). ( B ) Surface plasmon resonance sensorgrams showing change in response units (RU) caused by binding and dissociation of Fab fragments using different conformational states of TG2. When using TG2 covalently linked to an active-site inhibitor (iTG2) in the presence of Ca 2+ , only antibodies recognizing core-domain epitopes bound detectably, whereas all but one antibody showed binding in the presence of GDP. This antibody could still bind TG2 in the absence of effector molecules. ( C ) Comparison of TG2 affinities between Fab fragments specific to N-terminal ( n = 9) and non–N-terminal ( n = 7) epitopes. Plotted values were obtained with the TG2 conformation that, in each case, gave the highest affinity. Column heights indicate mean affinity, and statistical difference was evaluated using an unpaired t test. ( D ) Immunofluorescence detection of mAbs bound to human recombinant TG3/TG2, which was immobilized on mouse TG2 KO small intestinal tissue sections. ( E ) Representative flow cytometry plot ( Left ) and mean distribution of TG2-reactive plasma cells (PCs) between populations targeting C2 and non-C2 epitopes in three celiac patients ( Right ). The two populations were identified by costaining with full-length TG2 and a truncated TG2 variant consisting of the C2 domain only. ( F ) Western blots showing cross-link formation between TG2 and a biotinylated 33mer gluten peptide resulting in high-molecular-weight bands. Cross-linked TG2-gluten complexes were immunoprecipitated (IP) with the indicated mAbs, and protein bands were visualized using either anti-TG2 mAb or streptavidin (SA).

    Techniques Used: Binding Assay, SPR Assay, Affinity Column, Immunofluorescence, Recombinant, Flow Cytometry, Cytometry, Variant Assay, Western Blot, Molecular Weight, Immunoprecipitation

    40) Product Images from "Activation of Pre-mRNA Splicing by Human RNPS1 Is Regulated by CK2 Phosphorylation †"

    Article Title: Activation of Pre-mRNA Splicing by Human RNPS1 Is Regulated by CK2 Phosphorylation †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.4.1446-1457.2005

    Analyses of spliceosome complexes and RNA products by in vitro splicing assays with δ-crystallin pre-mRNA. (A) Schematic representation of the preparation of immobilized δ-crystallin pre-mRNA on streptavidin-agarose. B and AV indicate
    Figure Legend Snippet: Analyses of spliceosome complexes and RNA products by in vitro splicing assays with δ-crystallin pre-mRNA. (A) Schematic representation of the preparation of immobilized δ-crystallin pre-mRNA on streptavidin-agarose. B and AV indicate

    Techniques Used: In Vitro

    Related Articles

    Purification:

    Article Title: RNA affinity tags for purification of RNAs and ribonucleoprotein complexes
    Article Snippet: .. The amount of affinity matrix used to isolate the tagged complexes should really be determined experimentally, but as a rough guide to purification of complexes from crude yeast lysates, approximately 10–20 μl Sephadex G-200 (Pharmacia) or streptavidin–agarose (Sigma) is used for each milligram of protein in the lysates. .. The binding step is usually carried out at 4 °C for 1 h. For isolation of S1-tagged complexes, incubation of the lysates with egg white avidin before the binding step is recommended, especially for yeast lysates.

    Incubation:

    Article Title: Two new mutations in the HIF2A gene associated with erythrocytosis
    Article Snippet: .. Ten ng of recombinant VHL was incubated without or with 1 μg of biotinylated hydroxyproline (Hyp)-564 HIF-1α (556–574) prebound to 10 μl of streptavidin-agarose (Sigma) in the absence or presence of 5 nM wild type (WT) or F540L Hyp-531 HIF-2α (527–542) peptide. .. The resins were washed, eluted, and the eluates subjected to SDS-PAGE and western blotting using anti-Flag antibodies (Sigma), essentially as described [ , ].

    other:

    Article Title: INTRACELLULAR GLUTATHIONE MEDIATES THE DENITROSYLATION OF PROTEIN NITROSOTHIOLS IN THE RAT SPINAL CORD
    Article Snippet: Ascorbic acid, aurothioglucose (ATG), caffeic acid (CA), diethyl maleate (DEM), 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB), L-glutathione (GSH), L-glutathione monoethyl ester (GSH-EE), mefenamic acid (MEF), mercaptosuccinic acid (MSA), methyl methanethiol sulfonate, 4-methylpyrazole (4-MP), phenylarsine oxide (PAO) and streptavidin-agarose were purchased from Sigma-Aldrich (St. Louis, MO).

    Article Title: Lasiodin Inhibits Proliferation of Human Nasopharyngeal Carcinoma Cells by Simultaneous Modulation of the Apaf-1/Caspase, AKT/MAPK and COX-2/NF-?B Signaling Pathways
    Article Snippet: Reagents LY294002, SB203580, SP600125, U0126, celecoxib and streptavidin-agarose were purchased from Sigma (St. Louis, MO).

    Lysis:

    Article Title: XIAP upregulates expression of HIF target genes by targeting HIF1α for Lys63-linked polyubiquitination
    Article Snippet: .. Ubiquitination assays For ubiquitination assays, cells were lysed at room temperature under denaturing conditions (8 M urea, 50 mM Tris [pH 8.0], 300 mM NaCl, 50 mM Na2 HPO4 , 0.5% NP-40, 1 mM PMSF, supplemented with protease inhibitors) and ubiquitinated material was recovered by rotation with NiNTA-agarose (Invitrogen) or streptavidin-agarose (Sigma), washed 3× with lysis buffer and analysed by western blotting. .. Quantitative reverse transcription-PCR Total RNA was isolated using the Peqgold Total RNA Isolation Kit (Peqlab) according to the manufacturer's instructions.

    Western Blot:

    Article Title: XIAP upregulates expression of HIF target genes by targeting HIF1α for Lys63-linked polyubiquitination
    Article Snippet: .. Ubiquitination assays For ubiquitination assays, cells were lysed at room temperature under denaturing conditions (8 M urea, 50 mM Tris [pH 8.0], 300 mM NaCl, 50 mM Na2 HPO4 , 0.5% NP-40, 1 mM PMSF, supplemented with protease inhibitors) and ubiquitinated material was recovered by rotation with NiNTA-agarose (Invitrogen) or streptavidin-agarose (Sigma), washed 3× with lysis buffer and analysed by western blotting. .. Quantitative reverse transcription-PCR Total RNA was isolated using the Peqgold Total RNA Isolation Kit (Peqlab) according to the manufacturer's instructions.

    Article Title: The p29 and p35 Immunodominant Antigens of Neospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii
    Article Snippet: .. Alternatively, the biotin-labeled proteins were precipitated with streptavidin-agarose (Sigma) and analyzed by Western blotting, as described above, using the MAb 6C11 or 5H5. ..

    Article Title: Cell-surface Processing of the Metalloprotease Pro-ADAMTS9 Is Influenced by the Chaperone GRP94/gp96 *
    Article Snippet: .. Biotinylated proteins were captured using streptavidin-agarose (Sigma-Aldrich) and eluted by boiling in Laemmli sample buffer, followed by SDS-PAGE and Western blotting with appropriate antibodies. .. To detect possible biotinylation of intracellular proteins, as a control for validity of cell-surface biotinylation, Western blotting was done for the cell lysate or the biotinylated proteins with monoclonal anti-α-actinin-4 antibody.

    Recombinant:

    Article Title: Two new mutations in the HIF2A gene associated with erythrocytosis
    Article Snippet: .. Ten ng of recombinant VHL was incubated without or with 1 μg of biotinylated hydroxyproline (Hyp)-564 HIF-1α (556–574) prebound to 10 μl of streptavidin-agarose (Sigma) in the absence or presence of 5 nM wild type (WT) or F540L Hyp-531 HIF-2α (527–542) peptide. .. The resins were washed, eluted, and the eluates subjected to SDS-PAGE and western blotting using anti-Flag antibodies (Sigma), essentially as described [ , ].

    SDS Page:

    Article Title: Cell-surface Processing of the Metalloprotease Pro-ADAMTS9 Is Influenced by the Chaperone GRP94/gp96 *
    Article Snippet: .. Biotinylated proteins were captured using streptavidin-agarose (Sigma-Aldrich) and eluted by boiling in Laemmli sample buffer, followed by SDS-PAGE and Western blotting with appropriate antibodies. .. To detect possible biotinylation of intracellular proteins, as a control for validity of cell-surface biotinylation, Western blotting was done for the cell lysate or the biotinylated proteins with monoclonal anti-α-actinin-4 antibody.

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    Millipore streptavidin agarose beads
    The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with <t>streptavidin</t> beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.
    Streptavidin Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore streptavidin
    ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of <t>streptavidin</t> (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).
    Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore streptavidin sepharose affinity columns
    <t>Streptavidin-affinity-enriched</t> PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using <t>streptavidin—Sepharose</t> affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.
    Streptavidin Sepharose Affinity Columns, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

    Journal: Glycobiology

    Article Title: Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

    doi: 10.1093/glycob/cwr097

    Figure Lengend Snippet: The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

    Article Snippet: The cell extracts were centrifuged at 15,000 × g for 30 min and supernatants were incubated with 50 μL of streptavidin–agarose beads (Millipore) for 1 h at 4°C.

    Techniques: Expressing, Synthesized, Labeling, Radioactivity, Incubation, Immunoprecipitation, Electrophoresis, Autoradiography

    Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

    Journal: Glycobiology

    Article Title: Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

    doi: 10.1093/glycob/cwr097

    Figure Lengend Snippet: Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

    Article Snippet: The cell extracts were centrifuged at 15,000 × g for 30 min and supernatants were incubated with 50 μL of streptavidin–agarose beads (Millipore) for 1 h at 4°C.

    Techniques: Inhibition, Expressing, Labeling, Western Blot

    Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with streptavidin beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).

    Journal: Journal of Virology

    Article Title: Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

    doi: 10.1128/JVI.00812-17

    Figure Lengend Snippet: Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with streptavidin beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).

    Article Snippet: Streptavidin agarose beads (Millipore) were prewashed once with 1× mild buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2 ), and 15 μg of the WT or PPXY mutant eVP40 peptide was incubated with the prewashed streptavidin beads in 500 μl of 1× mild buffer for 1 h at 4°C with rocking.

    Techniques: Mutagenesis, Western Blot, Expressing

    ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of streptavidin (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).

    Journal: Nucleic Acids Research

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

    doi: 10.1093/nar/gkt315

    Figure Lengend Snippet: ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of streptavidin (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).

    Article Snippet: The volumes of the binding reaction without EGTA or streptavidin were adjusted by addition of deionized water (Millipore).

    Techniques: Binding Assay, Agarose Gel Electrophoresis, Incubation, Blocking Assay, Staining

    Streptavidin-affinity-enriched PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.

    Journal: PLoS ONE

    Article Title: Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

    doi: 10.1371/journal.pone.0133033

    Figure Lengend Snippet: Streptavidin-affinity-enriched PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.

    Article Snippet: Protein fractions were eluted by streptavidin—Sepharose affinity columns, desalted, concentrated using AmiconTM Ultrafree centrifugal filters (Millipore), and visualized using a FOCUS-FAST silver-stain kit.

    Techniques: Affinity Chromatography, Far Western Blot, Silver Staining