mouse anti cbd  (New England Biolabs)


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    New England Biolabs mouse anti cbd
    Mouse Anti Cbd, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti cbd  (New England Biolabs)


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    New England Biolabs mouse anti cbd
    Mouse Anti Cbd, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cbd  (New England Biolabs)


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    New England Biolabs cbd
    A. MDA-MB-231 cells were transfected with an expression vector for HA-Nrf2, along with an empty vector or a plasmid for ENC1-Myc, <t>Keap1-CBD,</t> GAN1-Myc or Sarcosin-Myc. Transfected cells were lysed at 48 hr post-transfection and cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc, anti-CBD and <t>anti-tubulin</t> <t>antibodies.</t> B. An expression vector for HA-Nrf2 or HA-SMN gene was transfected into MDA-MB-231 cells, along with an empty vector or an expression vector for ENC1-Myc. Cells were harvested and immunoblot analysis was performed. C. An expression vector for ENC1-Myc was transfected into MDA-MB-231 cells. Cells were left untreated or treated with 50 µM tBHQ for 16 hr before cell lysis. Cell lysates were subjected to immunoblot analysis (left panel). The intensities of the Nrf2 bands were quantified by the ChemiDoc XRS gel documentation system and Quantity One software from Bio-Rad (right panel). D. MDA-MB-231 cells were transfected with different amounts of an ENC1-Myc expression vector, along with an expression vector for HA-Nrf2. Cell lysates were subjected to immunoblot analysis with anti-HA and anti-Derlin-1 antibodies.
    Cbd, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level"

    Article Title: Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005492

    A. MDA-MB-231 cells were transfected with an expression vector for HA-Nrf2, along with an empty vector or a plasmid for ENC1-Myc, Keap1-CBD, GAN1-Myc or Sarcosin-Myc. Transfected cells were lysed at 48 hr post-transfection and cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc, anti-CBD and anti-tubulin antibodies. B. An expression vector for HA-Nrf2 or HA-SMN gene was transfected into MDA-MB-231 cells, along with an empty vector or an expression vector for ENC1-Myc. Cells were harvested and immunoblot analysis was performed. C. An expression vector for ENC1-Myc was transfected into MDA-MB-231 cells. Cells were left untreated or treated with 50 µM tBHQ for 16 hr before cell lysis. Cell lysates were subjected to immunoblot analysis (left panel). The intensities of the Nrf2 bands were quantified by the ChemiDoc XRS gel documentation system and Quantity One software from Bio-Rad (right panel). D. MDA-MB-231 cells were transfected with different amounts of an ENC1-Myc expression vector, along with an expression vector for HA-Nrf2. Cell lysates were subjected to immunoblot analysis with anti-HA and anti-Derlin-1 antibodies.
    Figure Legend Snippet: A. MDA-MB-231 cells were transfected with an expression vector for HA-Nrf2, along with an empty vector or a plasmid for ENC1-Myc, Keap1-CBD, GAN1-Myc or Sarcosin-Myc. Transfected cells were lysed at 48 hr post-transfection and cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc, anti-CBD and anti-tubulin antibodies. B. An expression vector for HA-Nrf2 or HA-SMN gene was transfected into MDA-MB-231 cells, along with an empty vector or an expression vector for ENC1-Myc. Cells were harvested and immunoblot analysis was performed. C. An expression vector for ENC1-Myc was transfected into MDA-MB-231 cells. Cells were left untreated or treated with 50 µM tBHQ for 16 hr before cell lysis. Cell lysates were subjected to immunoblot analysis (left panel). The intensities of the Nrf2 bands were quantified by the ChemiDoc XRS gel documentation system and Quantity One software from Bio-Rad (right panel). D. MDA-MB-231 cells were transfected with different amounts of an ENC1-Myc expression vector, along with an expression vector for HA-Nrf2. Cell lysates were subjected to immunoblot analysis with anti-HA and anti-Derlin-1 antibodies.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, Lysis, Software

    A. MDA-MB-231 cells transfected with plasmids for ARE-driven firefly luciferase, TK-renilla luciferase and the indicated genes were treated with 7.5 µM SF or 50 µM tBHQ for 16 hr prior to cell lysis. Firefly and renilla luciferase activities were measured. Means and standard deviations were calculated from three independent experiments. B. Small aliquots of the lysates were subjected to immunoblot analysis with anti-Nrf2 (left panel), anti-HA, anti-Myc, anti-CBD and anti-β-actin antibodies (right panel). C. An empty plasmid or a plasmid for either ENC1-Myc or Keap1 was transfected into MDA-MB-231 cells and cells were treated with tBHQ for 16 hr. At 48 hr post-transfection, the relative mRNA levels of Nrf2, NQO1, HO-1, ENC1 and Keap1 were determined by real-time PCR.
    Figure Legend Snippet: A. MDA-MB-231 cells transfected with plasmids for ARE-driven firefly luciferase, TK-renilla luciferase and the indicated genes were treated with 7.5 µM SF or 50 µM tBHQ for 16 hr prior to cell lysis. Firefly and renilla luciferase activities were measured. Means and standard deviations were calculated from three independent experiments. B. Small aliquots of the lysates were subjected to immunoblot analysis with anti-Nrf2 (left panel), anti-HA, anti-Myc, anti-CBD and anti-β-actin antibodies (right panel). C. An empty plasmid or a plasmid for either ENC1-Myc or Keap1 was transfected into MDA-MB-231 cells and cells were treated with tBHQ for 16 hr. At 48 hr post-transfection, the relative mRNA levels of Nrf2, NQO1, HO-1, ENC1 and Keap1 were determined by real-time PCR.

    Techniques Used: Transfection, Luciferase, Lysis, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction

    A. MDA-MB-231 cells were transfected with an empty vector or a vector for ENC1-Myc. Transfected cells were treated with proteasome inhibitors MG132 (M) (10 µM), clasto-lactacystin β-lactone (Lac) (10 µM) or epoxomicin (Ep) (1 µM), or lysosome inhibitors chloroquine (Ch) (50 µM) or ammonium chloride (Ac) (50 mM) for 4 hr before cells were lysed at 48 hr post-transfection. Cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc and anti-β-actin antibodies. B. MDA-MB-231 cells were transfected with HA-Nrf2 and with an empty vector or ENC1-Myc. Transfected cells were treated with proteasome and lysosome inhibitors as described above. Immunoblot analysis was performed. C. In vivo ubiquitination assay was performed in MDA-MB-231 cells transfected with plasmids for HA-Ub and Gal4-Neh2, along with either ENC1-Myc or Keap1-CBD. Transfected cells were treated with 10 µM MG132 for 4 hr prior to cell lysis. Cell lysates were denatured by heating and subjected to immunoprecipitation with anti-Gal4 antibodies. The precipitated protein complexes were subjected to immunoblot analysis with anti-HA antibodies for detecting ubiquitin-conjugated Gal4-Neh2 (top panel). Small aliquots of total cell lysates were immunoblotted with the indicated antibodies (bottom three panels).
    Figure Legend Snippet: A. MDA-MB-231 cells were transfected with an empty vector or a vector for ENC1-Myc. Transfected cells were treated with proteasome inhibitors MG132 (M) (10 µM), clasto-lactacystin β-lactone (Lac) (10 µM) or epoxomicin (Ep) (1 µM), or lysosome inhibitors chloroquine (Ch) (50 µM) or ammonium chloride (Ac) (50 mM) for 4 hr before cells were lysed at 48 hr post-transfection. Cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc and anti-β-actin antibodies. B. MDA-MB-231 cells were transfected with HA-Nrf2 and with an empty vector or ENC1-Myc. Transfected cells were treated with proteasome and lysosome inhibitors as described above. Immunoblot analysis was performed. C. In vivo ubiquitination assay was performed in MDA-MB-231 cells transfected with plasmids for HA-Ub and Gal4-Neh2, along with either ENC1-Myc or Keap1-CBD. Transfected cells were treated with 10 µM MG132 for 4 hr prior to cell lysis. Cell lysates were denatured by heating and subjected to immunoprecipitation with anti-Gal4 antibodies. The precipitated protein complexes were subjected to immunoblot analysis with anti-HA antibodies for detecting ubiquitin-conjugated Gal4-Neh2 (top panel). Small aliquots of total cell lysates were immunoblotted with the indicated antibodies (bottom three panels).

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, In Vivo, Ubiquitin Assay, Lysis, Immunoprecipitation

    A. MDA-MB-231 cells were transfected with an expression vector for ENC1-Myc, along with an empty vector or an expression vector for Keap1-CBD. Cell lysates were subjected to chitin pull-down assay. Precipitated proteins were subjected to immunoblot analysis with anti-Myc and anti-CBD antibodies for detection of Keap1-CBD and ENC1-Myc (top panel). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (bottom panel). B. Cell lysates from MDA-MB-231 cells were immunoprecipitated with anti-Keap1 antibody. The precipitated protein complexes were subjected to immunoblot analysis with anti-Nrf2 and anti-ENC1 antibodies. IgG was included in the immunoprecipitation analysis as a negative control. C. Plasmids for full-length ENC1 (E-FL) and two ENC1 deletion mutants E320 and E570, Nrf2 and Luciferase (Luc) were used for in vitro transcription/translation to synthesize [ 35 S]-labeled proteins. The proteins were incubated with Keap1-His purified from E. Coli . Protein complexes containing Keap1 were pulled-down with nickel beads and resolved in SDS-PAGE and detected by autoradiography. Nrf2 and Luciferase were used as a positive and a negative control. 20% of [ 35 S]-labeled proteins were resolved by SDS-PAGE gel to show equivalent input of each protein (right panel). D. Keap1-/- and wild-type MEF cells were transfected with plasmids for HA-Nrf2 and ENC1-Myc. Cells were lysed at 48 hr post-transfection. Cell lysates were analyzed by immunoblot with anti-HA, anti-Myc and anti-β-actin. E. Cell lysates from MDA-MB-231 cells transfected with plasmids for Keap1-CBD, HA-Nrf2, and ENC1-Myc were used for pull-down assay with chitin beads Precipitated proteins were subjected to immunoblot analysis with anti-HA and anti-CBD antibodies (left figure, top two panels). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (left figure, bottom four panels). Nrf2 and β-Actin band intensities were quantified using Quantity One (BIO-RAD). The intensity of Nrf2 was normalized to that of β-actin (right graph).
    Figure Legend Snippet: A. MDA-MB-231 cells were transfected with an expression vector for ENC1-Myc, along with an empty vector or an expression vector for Keap1-CBD. Cell lysates were subjected to chitin pull-down assay. Precipitated proteins were subjected to immunoblot analysis with anti-Myc and anti-CBD antibodies for detection of Keap1-CBD and ENC1-Myc (top panel). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (bottom panel). B. Cell lysates from MDA-MB-231 cells were immunoprecipitated with anti-Keap1 antibody. The precipitated protein complexes were subjected to immunoblot analysis with anti-Nrf2 and anti-ENC1 antibodies. IgG was included in the immunoprecipitation analysis as a negative control. C. Plasmids for full-length ENC1 (E-FL) and two ENC1 deletion mutants E320 and E570, Nrf2 and Luciferase (Luc) were used for in vitro transcription/translation to synthesize [ 35 S]-labeled proteins. The proteins were incubated with Keap1-His purified from E. Coli . Protein complexes containing Keap1 were pulled-down with nickel beads and resolved in SDS-PAGE and detected by autoradiography. Nrf2 and Luciferase were used as a positive and a negative control. 20% of [ 35 S]-labeled proteins were resolved by SDS-PAGE gel to show equivalent input of each protein (right panel). D. Keap1-/- and wild-type MEF cells were transfected with plasmids for HA-Nrf2 and ENC1-Myc. Cells were lysed at 48 hr post-transfection. Cell lysates were analyzed by immunoblot with anti-HA, anti-Myc and anti-β-actin. E. Cell lysates from MDA-MB-231 cells transfected with plasmids for Keap1-CBD, HA-Nrf2, and ENC1-Myc were used for pull-down assay with chitin beads Precipitated proteins were subjected to immunoblot analysis with anti-HA and anti-CBD antibodies (left figure, top two panels). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (left figure, bottom four panels). Nrf2 and β-Actin band intensities were quantified using Quantity One (BIO-RAD). The intensity of Nrf2 was normalized to that of β-actin (right graph).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Pull Down Assay, Western Blot, Immunoprecipitation, Negative Control, Luciferase, In Vitro, Labeling, Incubation, Purification, SDS Page, Autoradiography

    domain cbd  (New England Biolabs)


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    New England Biolabs domain cbd
    (A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and <t>CBD-tagged</t> Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and <t>anti-CBD</t> <t>antibodies.</t> CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.
    Domain Cbd, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response"

    Article Title: Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006588

    (A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and CBD-tagged Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and anti-CBD antibodies. CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.
    Figure Legend Snippet: (A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and CBD-tagged Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and anti-CBD antibodies. CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.

    Techniques Used: Expressing, Western Blot, Binding Assay, Mutagenesis, Transfection, Immunofluorescence, Staining, Fluorescence, Microscopy

    mouse monoclonal chitin binding domain  (New England Biolabs)


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    New England Biolabs mouse monoclonal chitin binding domain
    Mouse Monoclonal Chitin Binding Domain, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cbd  (New England Biolabs)


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    New England Biolabs cbd
    (A-C) Flag-tagged STAT1 (A) or IRF9 (C) or <t>CBD-tagged</t> STAT2 (B) were expressed in 293T cells transfected with the respective expression plasmids and either vIRF-1 (virf1) or empty (-) expression vector; replicates were either left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h. Flag/CBD-tagged proteins were then immuno/affinity-precipitated from cell lysates, and coprecipitated ISGF3 proteins were detected <t>by</t> <t>immunoblotting.</t> Diagrams below the panels illustrate the main findings from each experiment. (D-E) Serial coprecipitations of STAT1 and STAT2 (D), STAT1 and IRF9 (E), and IRF9 and STAT2 (F), respectively Flag- and CBD-tagged, from lysates of transfected 293T cells expressing vIRF-1 (virf1) or cotransfected with empty vector (-). All transfectants were treated with IFNβ for 24 h prior to harvesting. First (Flag IP) and second (CBD AP) precipitates (Precip. 1, Precip. 2) were analyzed by immunoblotting for the tagged “bait” proteins, the third (endogenous) ISGF3 protein (including phosphorylated and total STAT1 and STAT2), and vIRF-1; lysates were immunoblotted for expression of input proteins. Illustrated below each panel of blots are the main findings. (G) Disruption by vIRF-1 of STAT1-STAT2 complexes isolated by Flag-IP (STAT1) and CBD-AP (STAT2) from IFNβ-treated transfected 293T cells. Immunoprecipitated material from vIRF-1-Flag (virf1) or empty control (cntl) vector-transfected cells was applied in two concentrations (1x, 2x) to dual-precipitation-derived STAT1/STAT2 complexes, and then mixtures were subjected to re-precipitation with chitin beads (binding STAT2-CBD). STAT1 and vIRF-1 associated with re-precipitated STAT2-CBD were identified by immunoblotting. Relative levels of co-precipitated STAT1, normalized to affinity-sedimented STAT2, are shown below the STAT1 blot (cntl/1x value set at 1). (H) An equivalent experiment was carried out using GST-fused recombinant vIRF-1 (virf1) or GST (negative control) to challenge STAT1 interaction with STAT2 in STAT1/STAT2 hetero-complexes isolated by IP/AP dual precipitations from IFNβ-treated 293T cells. Endogenous IRF9 interaction with STAT1/2 and competition by vIRF-1 were also monitored. Relative levels and integrities of the recombinant proteins are shown in the Coomassie-stained gel (right); arrowheads indicate the positions of the full-length proteins.
    Cbd, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling"

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1010676

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

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

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

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

    gapdh rabbit monoclonal  (New England Biolabs)


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    New England Biolabs gapdh rabbit monoclonal
    a Volcano Plot highlighting the most differentially expressed genes in Z46 compared to controls following DESeq analysis. b RT-qPCR reveals mRNA expression levels for the targeted genes: MLIP 5–6 accounts specifically for transcripts containing the nonsense mutation, MLIP 9–10 covers all the known transcripts, <t>and</t> <t>LMNA</t> probe accounts for both lamin A and C. RPS29 is used for data normalization, error bars correspond to standard deviation (triplicata). c, d Western Blot analysis reveals the presence of four isoforms of MLIP, two of which can be quantified (27 kDa and 50 kDa). Both lamin types also appear for LMNA and were analyzed separately. <t>GAPDH</t> is used as the reference gene for normalization of the blot in both instances.
    Gapdh Rabbit Monoclonal, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Novel homozygous nonsense mutation of MLIP and compensatory alternative splicing"

    Article Title: Novel homozygous nonsense mutation of MLIP and compensatory alternative splicing

    Journal: NPJ Genomic Medicine

    doi: 10.1038/s41525-022-00307-y

    a Volcano Plot highlighting the most differentially expressed genes in Z46 compared to controls following DESeq analysis. b RT-qPCR reveals mRNA expression levels for the targeted genes: MLIP 5–6 accounts specifically for transcripts containing the nonsense mutation, MLIP 9–10 covers all the known transcripts, and LMNA probe accounts for both lamin A and C. RPS29 is used for data normalization, error bars correspond to standard deviation (triplicata). c, d Western Blot analysis reveals the presence of four isoforms of MLIP, two of which can be quantified (27 kDa and 50 kDa). Both lamin types also appear for LMNA and were analyzed separately. GAPDH is used as the reference gene for normalization of the blot in both instances.
    Figure Legend Snippet: a Volcano Plot highlighting the most differentially expressed genes in Z46 compared to controls following DESeq analysis. b RT-qPCR reveals mRNA expression levels for the targeted genes: MLIP 5–6 accounts specifically for transcripts containing the nonsense mutation, MLIP 9–10 covers all the known transcripts, and LMNA probe accounts for both lamin A and C. RPS29 is used for data normalization, error bars correspond to standard deviation (triplicata). c, d Western Blot analysis reveals the presence of four isoforms of MLIP, two of which can be quantified (27 kDa and 50 kDa). Both lamin types also appear for LMNA and were analyzed separately. GAPDH is used as the reference gene for normalization of the blot in both instances.

    Techniques Used: Quantitative RT-PCR, Expressing, Mutagenesis, Standard Deviation, Western Blot

    mouse anti cbd monoclonal antibody  (New England Biolabs)


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    New England Biolabs mouse anti cbd monoclonal antibody
    Mouse Anti Cbd Monoclonal Antibody, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    e80345  (New England Biolabs)


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    New England Biolabs e80345
    Antibodies
    E80345, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway"

    Article Title: Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202010179

    Antibodies
    Figure Legend Snippet: Antibodies

    Techniques Used: Western Blot, Proximity Ligation Assay

    rabbit monoclonal  (New England Biolabs)


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    New England Biolabs rabbit monoclonal
    Rabbit Monoclonal, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti pn peptide  (New England Biolabs)


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    New England Biolabs anti pn peptide
    Anti Pn Peptide, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs mouse anti cbd
    Mouse Anti Cbd, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs cbd
    A. MDA-MB-231 cells were transfected with an expression vector for HA-Nrf2, along with an empty vector or a plasmid for ENC1-Myc, <t>Keap1-CBD,</t> GAN1-Myc or Sarcosin-Myc. Transfected cells were lysed at 48 hr post-transfection and cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc, anti-CBD and <t>anti-tubulin</t> <t>antibodies.</t> B. An expression vector for HA-Nrf2 or HA-SMN gene was transfected into MDA-MB-231 cells, along with an empty vector or an expression vector for ENC1-Myc. Cells were harvested and immunoblot analysis was performed. C. An expression vector for ENC1-Myc was transfected into MDA-MB-231 cells. Cells were left untreated or treated with 50 µM tBHQ for 16 hr before cell lysis. Cell lysates were subjected to immunoblot analysis (left panel). The intensities of the Nrf2 bands were quantified by the ChemiDoc XRS gel documentation system and Quantity One software from Bio-Rad (right panel). D. MDA-MB-231 cells were transfected with different amounts of an ENC1-Myc expression vector, along with an expression vector for HA-Nrf2. Cell lysates were subjected to immunoblot analysis with anti-HA and anti-Derlin-1 antibodies.
    Cbd, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and <t>CBD-tagged</t> Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and <t>anti-CBD</t> <t>antibodies.</t> CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.
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    New England Biolabs mouse monoclonal chitin binding domain
    (A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and <t>CBD-tagged</t> Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and <t>anti-CBD</t> <t>antibodies.</t> CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.
    Mouse Monoclonal Chitin Binding Domain, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs gapdh rabbit monoclonal
    a Volcano Plot highlighting the most differentially expressed genes in Z46 compared to controls following DESeq analysis. b RT-qPCR reveals mRNA expression levels for the targeted genes: MLIP 5–6 accounts specifically for transcripts containing the nonsense mutation, MLIP 9–10 covers all the known transcripts, <t>and</t> <t>LMNA</t> probe accounts for both lamin A and C. RPS29 is used for data normalization, error bars correspond to standard deviation (triplicata). c, d Western Blot analysis reveals the presence of four isoforms of MLIP, two of which can be quantified (27 kDa and 50 kDa). Both lamin types also appear for LMNA and were analyzed separately. <t>GAPDH</t> is used as the reference gene for normalization of the blot in both instances.
    Gapdh Rabbit Monoclonal, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs mouse anti cbd monoclonal antibody
    a Volcano Plot highlighting the most differentially expressed genes in Z46 compared to controls following DESeq analysis. b RT-qPCR reveals mRNA expression levels for the targeted genes: MLIP 5–6 accounts specifically for transcripts containing the nonsense mutation, MLIP 9–10 covers all the known transcripts, <t>and</t> <t>LMNA</t> probe accounts for both lamin A and C. RPS29 is used for data normalization, error bars correspond to standard deviation (triplicata). c, d Western Blot analysis reveals the presence of four isoforms of MLIP, two of which can be quantified (27 kDa and 50 kDa). Both lamin types also appear for LMNA and were analyzed separately. <t>GAPDH</t> is used as the reference gene for normalization of the blot in both instances.
    Mouse Anti Cbd Monoclonal Antibody, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs e80345
    Antibodies
    E80345, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rabbit monoclonal
    Antibodies
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    A. MDA-MB-231 cells were transfected with an expression vector for HA-Nrf2, along with an empty vector or a plasmid for ENC1-Myc, Keap1-CBD, GAN1-Myc or Sarcosin-Myc. Transfected cells were lysed at 48 hr post-transfection and cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc, anti-CBD and anti-tubulin antibodies. B. An expression vector for HA-Nrf2 or HA-SMN gene was transfected into MDA-MB-231 cells, along with an empty vector or an expression vector for ENC1-Myc. Cells were harvested and immunoblot analysis was performed. C. An expression vector for ENC1-Myc was transfected into MDA-MB-231 cells. Cells were left untreated or treated with 50 µM tBHQ for 16 hr before cell lysis. Cell lysates were subjected to immunoblot analysis (left panel). The intensities of the Nrf2 bands were quantified by the ChemiDoc XRS gel documentation system and Quantity One software from Bio-Rad (right panel). D. MDA-MB-231 cells were transfected with different amounts of an ENC1-Myc expression vector, along with an expression vector for HA-Nrf2. Cell lysates were subjected to immunoblot analysis with anti-HA and anti-Derlin-1 antibodies.

    Journal: PLoS ONE

    Article Title: Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level

    doi: 10.1371/journal.pone.0005492

    Figure Lengend Snippet: A. MDA-MB-231 cells were transfected with an expression vector for HA-Nrf2, along with an empty vector or a plasmid for ENC1-Myc, Keap1-CBD, GAN1-Myc or Sarcosin-Myc. Transfected cells were lysed at 48 hr post-transfection and cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc, anti-CBD and anti-tubulin antibodies. B. An expression vector for HA-Nrf2 or HA-SMN gene was transfected into MDA-MB-231 cells, along with an empty vector or an expression vector for ENC1-Myc. Cells were harvested and immunoblot analysis was performed. C. An expression vector for ENC1-Myc was transfected into MDA-MB-231 cells. Cells were left untreated or treated with 50 µM tBHQ for 16 hr before cell lysis. Cell lysates were subjected to immunoblot analysis (left panel). The intensities of the Nrf2 bands were quantified by the ChemiDoc XRS gel documentation system and Quantity One software from Bio-Rad (right panel). D. MDA-MB-231 cells were transfected with different amounts of an ENC1-Myc expression vector, along with an expression vector for HA-Nrf2. Cell lysates were subjected to immunoblot analysis with anti-HA and anti-Derlin-1 antibodies.

    Article Snippet: Antibodies against Nrf2, Myc, HA, β-Actin, Tubulin, Gal4 (Santa Cruz Biotechnology), and CBD (New England Biolabs) were purchased from commercial sources.

    Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Lysis, Software

    A. MDA-MB-231 cells transfected with plasmids for ARE-driven firefly luciferase, TK-renilla luciferase and the indicated genes were treated with 7.5 µM SF or 50 µM tBHQ for 16 hr prior to cell lysis. Firefly and renilla luciferase activities were measured. Means and standard deviations were calculated from three independent experiments. B. Small aliquots of the lysates were subjected to immunoblot analysis with anti-Nrf2 (left panel), anti-HA, anti-Myc, anti-CBD and anti-β-actin antibodies (right panel). C. An empty plasmid or a plasmid for either ENC1-Myc or Keap1 was transfected into MDA-MB-231 cells and cells were treated with tBHQ for 16 hr. At 48 hr post-transfection, the relative mRNA levels of Nrf2, NQO1, HO-1, ENC1 and Keap1 were determined by real-time PCR.

    Journal: PLoS ONE

    Article Title: Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level

    doi: 10.1371/journal.pone.0005492

    Figure Lengend Snippet: A. MDA-MB-231 cells transfected with plasmids for ARE-driven firefly luciferase, TK-renilla luciferase and the indicated genes were treated with 7.5 µM SF or 50 µM tBHQ for 16 hr prior to cell lysis. Firefly and renilla luciferase activities were measured. Means and standard deviations were calculated from three independent experiments. B. Small aliquots of the lysates were subjected to immunoblot analysis with anti-Nrf2 (left panel), anti-HA, anti-Myc, anti-CBD and anti-β-actin antibodies (right panel). C. An empty plasmid or a plasmid for either ENC1-Myc or Keap1 was transfected into MDA-MB-231 cells and cells were treated with tBHQ for 16 hr. At 48 hr post-transfection, the relative mRNA levels of Nrf2, NQO1, HO-1, ENC1 and Keap1 were determined by real-time PCR.

    Article Snippet: Antibodies against Nrf2, Myc, HA, β-Actin, Tubulin, Gal4 (Santa Cruz Biotechnology), and CBD (New England Biolabs) were purchased from commercial sources.

    Techniques: Transfection, Luciferase, Lysis, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction

    A. MDA-MB-231 cells were transfected with an empty vector or a vector for ENC1-Myc. Transfected cells were treated with proteasome inhibitors MG132 (M) (10 µM), clasto-lactacystin β-lactone (Lac) (10 µM) or epoxomicin (Ep) (1 µM), or lysosome inhibitors chloroquine (Ch) (50 µM) or ammonium chloride (Ac) (50 mM) for 4 hr before cells were lysed at 48 hr post-transfection. Cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc and anti-β-actin antibodies. B. MDA-MB-231 cells were transfected with HA-Nrf2 and with an empty vector or ENC1-Myc. Transfected cells were treated with proteasome and lysosome inhibitors as described above. Immunoblot analysis was performed. C. In vivo ubiquitination assay was performed in MDA-MB-231 cells transfected with plasmids for HA-Ub and Gal4-Neh2, along with either ENC1-Myc or Keap1-CBD. Transfected cells were treated with 10 µM MG132 for 4 hr prior to cell lysis. Cell lysates were denatured by heating and subjected to immunoprecipitation with anti-Gal4 antibodies. The precipitated protein complexes were subjected to immunoblot analysis with anti-HA antibodies for detecting ubiquitin-conjugated Gal4-Neh2 (top panel). Small aliquots of total cell lysates were immunoblotted with the indicated antibodies (bottom three panels).

    Journal: PLoS ONE

    Article Title: Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level

    doi: 10.1371/journal.pone.0005492

    Figure Lengend Snippet: A. MDA-MB-231 cells were transfected with an empty vector or a vector for ENC1-Myc. Transfected cells were treated with proteasome inhibitors MG132 (M) (10 µM), clasto-lactacystin β-lactone (Lac) (10 µM) or epoxomicin (Ep) (1 µM), or lysosome inhibitors chloroquine (Ch) (50 µM) or ammonium chloride (Ac) (50 mM) for 4 hr before cells were lysed at 48 hr post-transfection. Cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc and anti-β-actin antibodies. B. MDA-MB-231 cells were transfected with HA-Nrf2 and with an empty vector or ENC1-Myc. Transfected cells were treated with proteasome and lysosome inhibitors as described above. Immunoblot analysis was performed. C. In vivo ubiquitination assay was performed in MDA-MB-231 cells transfected with plasmids for HA-Ub and Gal4-Neh2, along with either ENC1-Myc or Keap1-CBD. Transfected cells were treated with 10 µM MG132 for 4 hr prior to cell lysis. Cell lysates were denatured by heating and subjected to immunoprecipitation with anti-Gal4 antibodies. The precipitated protein complexes were subjected to immunoblot analysis with anti-HA antibodies for detecting ubiquitin-conjugated Gal4-Neh2 (top panel). Small aliquots of total cell lysates were immunoblotted with the indicated antibodies (bottom three panels).

    Article Snippet: Antibodies against Nrf2, Myc, HA, β-Actin, Tubulin, Gal4 (Santa Cruz Biotechnology), and CBD (New England Biolabs) were purchased from commercial sources.

    Techniques: Transfection, Plasmid Preparation, Western Blot, In Vivo, Ubiquitin Assay, Lysis, Immunoprecipitation

    A. MDA-MB-231 cells were transfected with an expression vector for ENC1-Myc, along with an empty vector or an expression vector for Keap1-CBD. Cell lysates were subjected to chitin pull-down assay. Precipitated proteins were subjected to immunoblot analysis with anti-Myc and anti-CBD antibodies for detection of Keap1-CBD and ENC1-Myc (top panel). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (bottom panel). B. Cell lysates from MDA-MB-231 cells were immunoprecipitated with anti-Keap1 antibody. The precipitated protein complexes were subjected to immunoblot analysis with anti-Nrf2 and anti-ENC1 antibodies. IgG was included in the immunoprecipitation analysis as a negative control. C. Plasmids for full-length ENC1 (E-FL) and two ENC1 deletion mutants E320 and E570, Nrf2 and Luciferase (Luc) were used for in vitro transcription/translation to synthesize [ 35 S]-labeled proteins. The proteins were incubated with Keap1-His purified from E. Coli . Protein complexes containing Keap1 were pulled-down with nickel beads and resolved in SDS-PAGE and detected by autoradiography. Nrf2 and Luciferase were used as a positive and a negative control. 20% of [ 35 S]-labeled proteins were resolved by SDS-PAGE gel to show equivalent input of each protein (right panel). D. Keap1-/- and wild-type MEF cells were transfected with plasmids for HA-Nrf2 and ENC1-Myc. Cells were lysed at 48 hr post-transfection. Cell lysates were analyzed by immunoblot with anti-HA, anti-Myc and anti-β-actin. E. Cell lysates from MDA-MB-231 cells transfected with plasmids for Keap1-CBD, HA-Nrf2, and ENC1-Myc were used for pull-down assay with chitin beads Precipitated proteins were subjected to immunoblot analysis with anti-HA and anti-CBD antibodies (left figure, top two panels). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (left figure, bottom four panels). Nrf2 and β-Actin band intensities were quantified using Quantity One (BIO-RAD). The intensity of Nrf2 was normalized to that of β-actin (right graph).

    Journal: PLoS ONE

    Article Title: Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level

    doi: 10.1371/journal.pone.0005492

    Figure Lengend Snippet: A. MDA-MB-231 cells were transfected with an expression vector for ENC1-Myc, along with an empty vector or an expression vector for Keap1-CBD. Cell lysates were subjected to chitin pull-down assay. Precipitated proteins were subjected to immunoblot analysis with anti-Myc and anti-CBD antibodies for detection of Keap1-CBD and ENC1-Myc (top panel). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (bottom panel). B. Cell lysates from MDA-MB-231 cells were immunoprecipitated with anti-Keap1 antibody. The precipitated protein complexes were subjected to immunoblot analysis with anti-Nrf2 and anti-ENC1 antibodies. IgG was included in the immunoprecipitation analysis as a negative control. C. Plasmids for full-length ENC1 (E-FL) and two ENC1 deletion mutants E320 and E570, Nrf2 and Luciferase (Luc) were used for in vitro transcription/translation to synthesize [ 35 S]-labeled proteins. The proteins were incubated with Keap1-His purified from E. Coli . Protein complexes containing Keap1 were pulled-down with nickel beads and resolved in SDS-PAGE and detected by autoradiography. Nrf2 and Luciferase were used as a positive and a negative control. 20% of [ 35 S]-labeled proteins were resolved by SDS-PAGE gel to show equivalent input of each protein (right panel). D. Keap1-/- and wild-type MEF cells were transfected with plasmids for HA-Nrf2 and ENC1-Myc. Cells were lysed at 48 hr post-transfection. Cell lysates were analyzed by immunoblot with anti-HA, anti-Myc and anti-β-actin. E. Cell lysates from MDA-MB-231 cells transfected with plasmids for Keap1-CBD, HA-Nrf2, and ENC1-Myc were used for pull-down assay with chitin beads Precipitated proteins were subjected to immunoblot analysis with anti-HA and anti-CBD antibodies (left figure, top two panels). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (left figure, bottom four panels). Nrf2 and β-Actin band intensities were quantified using Quantity One (BIO-RAD). The intensity of Nrf2 was normalized to that of β-actin (right graph).

    Article Snippet: Antibodies against Nrf2, Myc, HA, β-Actin, Tubulin, Gal4 (Santa Cruz Biotechnology), and CBD (New England Biolabs) were purchased from commercial sources.

    Techniques: Transfection, Expressing, Plasmid Preparation, Pull Down Assay, Western Blot, Immunoprecipitation, Negative Control, Luciferase, In Vitro, Labeling, Incubation, Purification, SDS Page, Autoradiography

    (A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and CBD-tagged Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and anti-CBD antibodies. CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.

    Journal: PLoS ONE

    Article Title: Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response

    doi: 10.1371/journal.pone.0006588

    Figure Lengend Snippet: (A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and CBD-tagged Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and anti-CBD antibodies. CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.

    Article Snippet: Antibodies against the HA epitope (Santa Cruz), chitin-binding domain (CBD) (New England Biolab), α-tubulin (Santa Cruz), and phosphorylated serine/threonine followed by a proline (pS/TP) (Abcam) were purchased from commercial sources.

    Techniques: Expressing, Western Blot, Binding Assay, Mutagenesis, Transfection, Immunofluorescence, Staining, Fluorescence, Microscopy

    a Volcano Plot highlighting the most differentially expressed genes in Z46 compared to controls following DESeq analysis. b RT-qPCR reveals mRNA expression levels for the targeted genes: MLIP 5–6 accounts specifically for transcripts containing the nonsense mutation, MLIP 9–10 covers all the known transcripts, and LMNA probe accounts for both lamin A and C. RPS29 is used for data normalization, error bars correspond to standard deviation (triplicata). c, d Western Blot analysis reveals the presence of four isoforms of MLIP, two of which can be quantified (27 kDa and 50 kDa). Both lamin types also appear for LMNA and were analyzed separately. GAPDH is used as the reference gene for normalization of the blot in both instances.

    Journal: NPJ Genomic Medicine

    Article Title: Novel homozygous nonsense mutation of MLIP and compensatory alternative splicing

    doi: 10.1038/s41525-022-00307-y

    Figure Lengend Snippet: a Volcano Plot highlighting the most differentially expressed genes in Z46 compared to controls following DESeq analysis. b RT-qPCR reveals mRNA expression levels for the targeted genes: MLIP 5–6 accounts specifically for transcripts containing the nonsense mutation, MLIP 9–10 covers all the known transcripts, and LMNA probe accounts for both lamin A and C. RPS29 is used for data normalization, error bars correspond to standard deviation (triplicata). c, d Western Blot analysis reveals the presence of four isoforms of MLIP, two of which can be quantified (27 kDa and 50 kDa). Both lamin types also appear for LMNA and were analyzed separately. GAPDH is used as the reference gene for normalization of the blot in both instances.

    Article Snippet: Antibodies used in this report: MLIP rabbit-polyclonal (Invitrogen PA5–72759, 1:2000 dilution); LMNA rabbit-polyclonal (NEB 2026 S, 1:1000 dilution); GAPDH rabbit-monoclonal (NEB 5174 S, 1:1000 dilution); HRP anti-rabbit (Jackson ImmunoResearch 111–035–114, 1:10000 dilution).

    Techniques: Quantitative RT-PCR, Expressing, Mutagenesis, Standard Deviation, Western Blot

    Antibodies

    Journal: The Journal of Cell Biology

    Article Title: Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway

    doi: 10.1083/jcb.202010179

    Figure Lengend Snippet: Antibodies

    Article Snippet: Anti-CBD , E80345 (New England Biolabs) , WB @ 1:800 , .

    Techniques: Western Blot, Proximity Ligation Assay