immunoprecipitation buffer  (Thermo Fisher)


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    Structured Review

    Thermo Fisher immunoprecipitation buffer
    p53 controls HIF-1α protein expression at 3 and 6 months after diabetes onset a, b The mRNA and protein expression of HIF-1α examined by qRT-PCR ( a ) and western blot ( b ) analysis of heart tissues at 3 and 6 months after diabetes onset. c Immunofluorescence staining using anti-HIF-1α antibody (red), DAPI for nuclei (blue), and phalloidin for actin fibers (green) of heart tissues at 6 months after diabetes onset (Scale bar = 25 µm). d A time course of 100 μM cycloheximide (CHX) treatment used to analyze the HIF-1α half-life in primary cardiomyocytes with or without PFT-α or p53-siRNA. β-actin used as loading control. (E-G) Protein-protein interaction between p53 and HIF-1α after PFT-α or p53-siRNA treatment in both primary cardiomyocytes e and heart tissues at 3 f and 6 g months after diabetes onset demonstrated by <t>immunoprecipitation</t> in the presence of the proteasomal inhibitor MG132. Data expressed as mean ± SD of three independent experiments. * P
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    Images

    1) Product Images from "Inhibition of p53 prevents diabetic cardiomyopathy by preventing early-stage apoptosis and cell senescence, reduced glycolysis, and impaired angiogenesis"

    Article Title: Inhibition of p53 prevents diabetic cardiomyopathy by preventing early-stage apoptosis and cell senescence, reduced glycolysis, and impaired angiogenesis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0093-5

    p53 controls HIF-1α protein expression at 3 and 6 months after diabetes onset a, b The mRNA and protein expression of HIF-1α examined by qRT-PCR ( a ) and western blot ( b ) analysis of heart tissues at 3 and 6 months after diabetes onset. c Immunofluorescence staining using anti-HIF-1α antibody (red), DAPI for nuclei (blue), and phalloidin for actin fibers (green) of heart tissues at 6 months after diabetes onset (Scale bar = 25 µm). d A time course of 100 μM cycloheximide (CHX) treatment used to analyze the HIF-1α half-life in primary cardiomyocytes with or without PFT-α or p53-siRNA. β-actin used as loading control. (E-G) Protein-protein interaction between p53 and HIF-1α after PFT-α or p53-siRNA treatment in both primary cardiomyocytes e and heart tissues at 3 f and 6 g months after diabetes onset demonstrated by immunoprecipitation in the presence of the proteasomal inhibitor MG132. Data expressed as mean ± SD of three independent experiments. * P
    Figure Legend Snippet: p53 controls HIF-1α protein expression at 3 and 6 months after diabetes onset a, b The mRNA and protein expression of HIF-1α examined by qRT-PCR ( a ) and western blot ( b ) analysis of heart tissues at 3 and 6 months after diabetes onset. c Immunofluorescence staining using anti-HIF-1α antibody (red), DAPI for nuclei (blue), and phalloidin for actin fibers (green) of heart tissues at 6 months after diabetes onset (Scale bar = 25 µm). d A time course of 100 μM cycloheximide (CHX) treatment used to analyze the HIF-1α half-life in primary cardiomyocytes with or without PFT-α or p53-siRNA. β-actin used as loading control. (E-G) Protein-protein interaction between p53 and HIF-1α after PFT-α or p53-siRNA treatment in both primary cardiomyocytes e and heart tissues at 3 f and 6 g months after diabetes onset demonstrated by immunoprecipitation in the presence of the proteasomal inhibitor MG132. Data expressed as mean ± SD of three independent experiments. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Immunoprecipitation

    p53 facilitates HIF-1α ubiquitination and consequent proteasomal degradation in an MDM2-dependent manner a HIF-1α polyubiquitination examined by immunoprecipitation and western blot of primary cardiomyocytes in the presence of MG132 with or without PFT-α or p53-siRNA treatment. b The MDM2 protein expression detected by western blot analysis. c, d The protein-protein interaction between MDM2 and HIF-1α detected at 3 ( c ) and 6 ( d ) months after diabetes onset by immunoprecipitation in vivo. e The protein-protein interaction between MDM2 and HIF-1α detected by immunoprecipitation in primary cardiomyocytes after transfection with Ctrl-siRNA or MDM2-siRNA. f HIF-1α polyubiquitination examined by immunoprecipitation and western blot in primary cardiomyocytes after transfection with Ctrl-siRNA or MDM2-siRNA. Data expressed as mean ± SD of three independent experiments. * P
    Figure Legend Snippet: p53 facilitates HIF-1α ubiquitination and consequent proteasomal degradation in an MDM2-dependent manner a HIF-1α polyubiquitination examined by immunoprecipitation and western blot of primary cardiomyocytes in the presence of MG132 with or without PFT-α or p53-siRNA treatment. b The MDM2 protein expression detected by western blot analysis. c, d The protein-protein interaction between MDM2 and HIF-1α detected at 3 ( c ) and 6 ( d ) months after diabetes onset by immunoprecipitation in vivo. e The protein-protein interaction between MDM2 and HIF-1α detected by immunoprecipitation in primary cardiomyocytes after transfection with Ctrl-siRNA or MDM2-siRNA. f HIF-1α polyubiquitination examined by immunoprecipitation and western blot in primary cardiomyocytes after transfection with Ctrl-siRNA or MDM2-siRNA. Data expressed as mean ± SD of three independent experiments. * P

    Techniques Used: Immunoprecipitation, Western Blot, Expressing, In Vivo, Transfection

    2) Product Images from "Targeting an antimicrobial effector function in insect immunity as a pest control strategy"

    Article Title: Targeting an antimicrobial effector function in insect immunity as a pest control strategy

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

    doi: 10.1073/pnas.0904063106

    )-glucanase induced by pathogenic patterns. ( A ) Immunoprecipitation of tGNBP-2 from termite extracts, nests, unprocessed wood, and soils A and B sampled from different locations. ( B )-Glucanase activity
    Figure Legend Snippet: )-glucanase induced by pathogenic patterns. ( A ) Immunoprecipitation of tGNBP-2 from termite extracts, nests, unprocessed wood, and soils A and B sampled from different locations. ( B )-Glucanase activity

    Techniques Used: Immunoprecipitation, Activity Assay

    3) Product Images from "A Synthetic Antibody Fragment Targeting Nicastrin Affects Assembly and Trafficking of γ-Secretase *"

    Article Title: A Synthetic Antibody Fragment Targeting Nicastrin Affects Assembly and Trafficking of γ-Secretase *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.609636

    Generation and characterization of the NCT-specific single-chain variable fragment. A , immunoprecipitation of NCT full-length (ECD) or truncated (716) ectodomain with NCT-specific Fabs under native condition. Fabs 12, 2, A9, and G9 are NCT-specific Fabs.
    Figure Legend Snippet: Generation and characterization of the NCT-specific single-chain variable fragment. A , immunoprecipitation of NCT full-length (ECD) or truncated (716) ectodomain with NCT-specific Fabs under native condition. Fabs 12, 2, A9, and G9 are NCT-specific Fabs.

    Techniques Used: Immunoprecipitation

    4) Product Images from "Structural protein 4.1R is integrally involved in nuclear envelope protein localization, centrosome-nucleus association and transcriptional signaling"

    Article Title: Structural protein 4.1R is integrally involved in nuclear envelope protein localization, centrosome-nucleus association and transcriptional signaling

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.077883

    4.1R and emerin co-immunoprecipitate and partially colocalize in human and murine fibroblasts. ( A ) Immunoprecipitation (IP) from HeLa whole cell sonicates. Clarified whole cell sonicates were prepared, immunoprecipitated with anti-4.1R antibody, and eluted
    Figure Legend Snippet: 4.1R and emerin co-immunoprecipitate and partially colocalize in human and murine fibroblasts. ( A ) Immunoprecipitation (IP) from HeLa whole cell sonicates. Clarified whole cell sonicates were prepared, immunoprecipitated with anti-4.1R antibody, and eluted

    Techniques Used: Immunoprecipitation

    5) Product Images from "Expression of Multidrug Resistance Associated Protein 5 (MRP5) on Cornea and Its Role in Drug Efflux"

    Article Title: Expression of Multidrug Resistance Associated Protein 5 (MRP5) on Cornea and Its Role in Drug Efflux

    Journal: Journal of Ocular Pharmacology and Therapeutics

    doi: 10.1089/jop.2008.0084

    Immunoprecipitation followed by Western blot indicating the presence of MRP5 in membrane fraction of SV40-HCEC and MDCKII-MRP5 (+ve control).
    Figure Legend Snippet: Immunoprecipitation followed by Western blot indicating the presence of MRP5 in membrane fraction of SV40-HCEC and MDCKII-MRP5 (+ve control).

    Techniques Used: Immunoprecipitation, Western Blot

    6) Product Images from "Inhibition of p53 prevents diabetic cardiomyopathy by preventing early-stage apoptosis and cell senescence, reduced glycolysis, and impaired angiogenesis"

    Article Title: Inhibition of p53 prevents diabetic cardiomyopathy by preventing early-stage apoptosis and cell senescence, reduced glycolysis, and impaired angiogenesis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0093-5

    p53 controls HIF-1α protein expression at 3 and 6 months after diabetes onset a, b The mRNA and protein expression of HIF-1α examined by qRT-PCR ( a ) and western blot ( b ) analysis of heart tissues at 3 and 6 months after diabetes onset. c Immunofluorescence staining using anti-HIF-1α antibody (red), DAPI for nuclei (blue), and phalloidin for actin fibers (green) of heart tissues at 6 months after diabetes onset (Scale bar = 25 µm). d A time course of 100 μM cycloheximide (CHX) treatment used to analyze the HIF-1α half-life in primary cardiomyocytes with or without PFT-α or p53-siRNA. β-actin used as loading control. (E-G) Protein-protein interaction between p53 and HIF-1α after PFT-α or p53-siRNA treatment in both primary cardiomyocytes e and heart tissues at 3 f and 6 g months after diabetes onset demonstrated by immunoprecipitation in the presence of the proteasomal inhibitor MG132. Data expressed as mean ± SD of three independent experiments. * P
    Figure Legend Snippet: p53 controls HIF-1α protein expression at 3 and 6 months after diabetes onset a, b The mRNA and protein expression of HIF-1α examined by qRT-PCR ( a ) and western blot ( b ) analysis of heart tissues at 3 and 6 months after diabetes onset. c Immunofluorescence staining using anti-HIF-1α antibody (red), DAPI for nuclei (blue), and phalloidin for actin fibers (green) of heart tissues at 6 months after diabetes onset (Scale bar = 25 µm). d A time course of 100 μM cycloheximide (CHX) treatment used to analyze the HIF-1α half-life in primary cardiomyocytes with or without PFT-α or p53-siRNA. β-actin used as loading control. (E-G) Protein-protein interaction between p53 and HIF-1α after PFT-α or p53-siRNA treatment in both primary cardiomyocytes e and heart tissues at 3 f and 6 g months after diabetes onset demonstrated by immunoprecipitation in the presence of the proteasomal inhibitor MG132. Data expressed as mean ± SD of three independent experiments. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Immunoprecipitation

    p53 facilitates HIF-1α ubiquitination and consequent proteasomal degradation in an MDM2-dependent manner a HIF-1α polyubiquitination examined by immunoprecipitation and western blot of primary cardiomyocytes in the presence of MG132 with or without PFT-α or p53-siRNA treatment. b The MDM2 protein expression detected by western blot analysis. c, d The protein-protein interaction between MDM2 and HIF-1α detected at 3 ( c ) and 6 ( d ) months after diabetes onset by immunoprecipitation in vivo. e The protein-protein interaction between MDM2 and HIF-1α detected by immunoprecipitation in primary cardiomyocytes after transfection with Ctrl-siRNA or MDM2-siRNA. f HIF-1α polyubiquitination examined by immunoprecipitation and western blot in primary cardiomyocytes after transfection with Ctrl-siRNA or MDM2-siRNA. Data expressed as mean ± SD of three independent experiments. * P
    Figure Legend Snippet: p53 facilitates HIF-1α ubiquitination and consequent proteasomal degradation in an MDM2-dependent manner a HIF-1α polyubiquitination examined by immunoprecipitation and western blot of primary cardiomyocytes in the presence of MG132 with or without PFT-α or p53-siRNA treatment. b The MDM2 protein expression detected by western blot analysis. c, d The protein-protein interaction between MDM2 and HIF-1α detected at 3 ( c ) and 6 ( d ) months after diabetes onset by immunoprecipitation in vivo. e The protein-protein interaction between MDM2 and HIF-1α detected by immunoprecipitation in primary cardiomyocytes after transfection with Ctrl-siRNA or MDM2-siRNA. f HIF-1α polyubiquitination examined by immunoprecipitation and western blot in primary cardiomyocytes after transfection with Ctrl-siRNA or MDM2-siRNA. Data expressed as mean ± SD of three independent experiments. * P

    Techniques Used: Immunoprecipitation, Western Blot, Expressing, In Vivo, Transfection

    7) Product Images from "Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila"

    Article Title: Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.02064

    Cohesin binds multiple sites in the cut regulatory region in Kc cells. Chromatin immunoprecipitation was performed with pre-immune and immune serum for Smc1 and Stromalin, and PCR amplicons spaced 1 kbp apart starting 0.6 kbp upstream of the wing margin enhancer (salmon-colored bar) extending into the transcribed region (blue-green bar) 2.8 kbp downstream of the transcription start site. Enrichment of each amplicon is plotted as the ratio of the amount of PCR product obtained with the immune serum relative to the amount obtained with the pre-immune serum. Most points represent the average of two or three measurements. Enrichment by Smc1 immune serum is plotted in blue, enrichment by Stromalin serum in red, and the black line is the average of the Smc1 and Stromalin values. This reveals cohesin-binding sites at 0.5, 4, 30.5, and 68 kbp upstream of the promoter. These peaks are recognized by an increase in the immune to pre-immune ratio relative to the baseline, which as expected, is close to 1.
    Figure Legend Snippet: Cohesin binds multiple sites in the cut regulatory region in Kc cells. Chromatin immunoprecipitation was performed with pre-immune and immune serum for Smc1 and Stromalin, and PCR amplicons spaced 1 kbp apart starting 0.6 kbp upstream of the wing margin enhancer (salmon-colored bar) extending into the transcribed region (blue-green bar) 2.8 kbp downstream of the transcription start site. Enrichment of each amplicon is plotted as the ratio of the amount of PCR product obtained with the immune serum relative to the amount obtained with the pre-immune serum. Most points represent the average of two or three measurements. Enrichment by Smc1 immune serum is plotted in blue, enrichment by Stromalin serum in red, and the black line is the average of the Smc1 and Stromalin values. This reveals cohesin-binding sites at 0.5, 4, 30.5, and 68 kbp upstream of the promoter. These peaks are recognized by an increase in the immune to pre-immune ratio relative to the baseline, which as expected, is close to 1.

    Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Binding Assay

    8) Product Images from "Circadian-Related Heteromerization of Adrenergic and Dopamine D4 Receptors Modulates Melatonin Synthesis and Release in the Pineal GlandNovel Melatonin-Blocking Complex Helps Control Body Rhythms"

    Article Title: Circadian-Related Heteromerization of Adrenergic and Dopamine D4 Receptors Modulates Melatonin Synthesis and Release in the Pineal GlandNovel Melatonin-Blocking Complex Helps Control Body Rhythms

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001347

    D 4 receptors form heteromers with α 1B and β 1 receptors in the pineal gland. In (A to C), pinealocytes were isolated from pineal glands extracted at 9:00 h (top) or at 20:00 h (bottom) and stained using anti-S-arrestin antibody (green) and anti-D 4 (A), anti-α 1B (B), or anti-β 1 (C) antibodies (red) as indicated in Materials and Methods . Scale bar, 5 µm. In (D to F), pinealocytes were isolated from pineal glands extracted at 9:00 h (top) or at 20:00 h (bottom) and the expression of α 1B -D 4 (D) and β 1 -D 4 (E) receptor heteromers was visualized as punctate red fluorescent spots detected by confocal microscopy using the proximity ligation assay (see Materials and Methods ). Any expression of α 1B -β 1 receptor heteromers was seen (F). Scale bar, 20 µm. In (G and H), co-immunoprecipitation of D 4 and α 1B or D 4 and β 1 receptors from pineal gland extracted at 9:00 h (sunrise) or at 20:00 h (sunset) was performed. Glands were solubilized and processed for immunoprecipitation as described under Materials and Methods using goat anti-D 4 , rabbit anti-α 1 , or goat anti-β 1 receptor antibodies or goat anti-adenosine A 2B receptor antibody as a negative control (N.C.). Solubilized gland membranes (G) and immunoprecipitates (H) were analyzed by SDS-PAGE and immunoblotted using rabbit anti-α 1 , rabbit anti-β 1 receptor antibodies, or goat anti-β 1 receptor antibody. Immunoprecipitation experiments with anti-α 1 or anti-β 1 receptor antibodies (right image in H) were performed with pineal glands extracted at 9:00 h. IP, immunoprecipitation; WB, western blotting; MW, molecular mass.
    Figure Legend Snippet: D 4 receptors form heteromers with α 1B and β 1 receptors in the pineal gland. In (A to C), pinealocytes were isolated from pineal glands extracted at 9:00 h (top) or at 20:00 h (bottom) and stained using anti-S-arrestin antibody (green) and anti-D 4 (A), anti-α 1B (B), or anti-β 1 (C) antibodies (red) as indicated in Materials and Methods . Scale bar, 5 µm. In (D to F), pinealocytes were isolated from pineal glands extracted at 9:00 h (top) or at 20:00 h (bottom) and the expression of α 1B -D 4 (D) and β 1 -D 4 (E) receptor heteromers was visualized as punctate red fluorescent spots detected by confocal microscopy using the proximity ligation assay (see Materials and Methods ). Any expression of α 1B -β 1 receptor heteromers was seen (F). Scale bar, 20 µm. In (G and H), co-immunoprecipitation of D 4 and α 1B or D 4 and β 1 receptors from pineal gland extracted at 9:00 h (sunrise) or at 20:00 h (sunset) was performed. Glands were solubilized and processed for immunoprecipitation as described under Materials and Methods using goat anti-D 4 , rabbit anti-α 1 , or goat anti-β 1 receptor antibodies or goat anti-adenosine A 2B receptor antibody as a negative control (N.C.). Solubilized gland membranes (G) and immunoprecipitates (H) were analyzed by SDS-PAGE and immunoblotted using rabbit anti-α 1 , rabbit anti-β 1 receptor antibodies, or goat anti-β 1 receptor antibody. Immunoprecipitation experiments with anti-α 1 or anti-β 1 receptor antibodies (right image in H) were performed with pineal glands extracted at 9:00 h. IP, immunoprecipitation; WB, western blotting; MW, molecular mass.

    Techniques Used: Isolation, Staining, Expressing, Confocal Microscopy, Proximity Ligation Assay, Immunoprecipitation, Negative Control, SDS Page, Western Blot

    9) Product Images from "Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells"

    Article Title: Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201207047

    LOK and SLK are responsible for ezrin/radixin C-terminal phosphorylation in Jeg-3 cells. (A) Enrichment statistics for top four kinases identified by mass spectrometry analysis of ezrin-iFlag cross-linking immunoprecipitates. LOK and SLK were the most highly enriched serine/threonine kinases binding to ezrin-iFlag. (B) LOK-GFP was coexpressed with empty vector or ezrin-iFlag, and cells were subjected to cross-linking immunoprecipitation using anti-Flag (Flag IP). LOK-GFP coimmunoprecipitates with ezrin-iFlag. (C) Cells were treated with the indicated combinations of validated siRNAs against LOK (siLOK1), SLK (siSLK2), or control (siGL2) and Western blotted for LOK to measure knockdown efficiency. The level of phosphorylated ezrin and radixin was determined by Western blotting and quantified by densitometry. (D) Cells were treated with siLOK1 for 3 d and then fixed and stained for ezrin and F-actin. Knockdown of LOK causes loss of microvilli similar to the knockdown of ezrin ( Fig. 3 A ). (E) The presence of microvilli on cells treated with the indicated siRNA was assessed after ezrin and F-actin staining. (F) Cells were treated with DMSO, staurosporine, or erlotinib at the indicated concentrations for 5 min at 37°C, and pERM levels were measured by Western blotting. (G) Cells treated as in F were fixed and stained for ezrin and F-actin. Erlotinib treatment causes a severe reduction in ezrin-containing microvilli. (H) GFP-Flag–tagged kinase constructs were immunoprecipitated using the Flag tag after expression in HEK293T cells and then combined with purified GST-tagged ezrin C terminus in the presence of ATP, which was then subjected to pERM Western blotting. Both LOK and SLK kinase domains are able to phosphorylate the ezrin C-terminal tail in vitro. (I) Cells were cotransfected with an empty vector or GFP fusions of either LOK or SLK along with ezrin-iFlag. The cells were lysed, ezrin-iFlag was immunoprecipitated with anti-Flag, and the phosphorylation of ezrin-iFlag was detected by Western blotting. Both LOK-GFP and SLK-GFP overexpression cause an increase in the overall level of ezrin-iFlag phosphorylation. Data in C, E, and F are means ± SD of three independent experiments. WB, Western blot; Vec, vector. Bars, 10 µm.
    Figure Legend Snippet: LOK and SLK are responsible for ezrin/radixin C-terminal phosphorylation in Jeg-3 cells. (A) Enrichment statistics for top four kinases identified by mass spectrometry analysis of ezrin-iFlag cross-linking immunoprecipitates. LOK and SLK were the most highly enriched serine/threonine kinases binding to ezrin-iFlag. (B) LOK-GFP was coexpressed with empty vector or ezrin-iFlag, and cells were subjected to cross-linking immunoprecipitation using anti-Flag (Flag IP). LOK-GFP coimmunoprecipitates with ezrin-iFlag. (C) Cells were treated with the indicated combinations of validated siRNAs against LOK (siLOK1), SLK (siSLK2), or control (siGL2) and Western blotted for LOK to measure knockdown efficiency. The level of phosphorylated ezrin and radixin was determined by Western blotting and quantified by densitometry. (D) Cells were treated with siLOK1 for 3 d and then fixed and stained for ezrin and F-actin. Knockdown of LOK causes loss of microvilli similar to the knockdown of ezrin ( Fig. 3 A ). (E) The presence of microvilli on cells treated with the indicated siRNA was assessed after ezrin and F-actin staining. (F) Cells were treated with DMSO, staurosporine, or erlotinib at the indicated concentrations for 5 min at 37°C, and pERM levels were measured by Western blotting. (G) Cells treated as in F were fixed and stained for ezrin and F-actin. Erlotinib treatment causes a severe reduction in ezrin-containing microvilli. (H) GFP-Flag–tagged kinase constructs were immunoprecipitated using the Flag tag after expression in HEK293T cells and then combined with purified GST-tagged ezrin C terminus in the presence of ATP, which was then subjected to pERM Western blotting. Both LOK and SLK kinase domains are able to phosphorylate the ezrin C-terminal tail in vitro. (I) Cells were cotransfected with an empty vector or GFP fusions of either LOK or SLK along with ezrin-iFlag. The cells were lysed, ezrin-iFlag was immunoprecipitated with anti-Flag, and the phosphorylation of ezrin-iFlag was detected by Western blotting. Both LOK-GFP and SLK-GFP overexpression cause an increase in the overall level of ezrin-iFlag phosphorylation. Data in C, E, and F are means ± SD of three independent experiments. WB, Western blot; Vec, vector. Bars, 10 µm.

    Techniques Used: Mass Spectrometry, Binding Assay, Plasmid Preparation, Cross-linking Immunoprecipitation, Western Blot, Staining, Construct, Immunoprecipitation, FLAG-tag, Expressing, Purification, In Vitro, Over Expression

    10) Product Images from "The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch"

    Article Title: The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.036491

    HECTD1 binds to RARA and influences its ubiquitination. (A) HA-RARA(281-462), binds to Myc-HECTD1(1-551) expressed in rabbit reticulocyte lysates. Proteins were immunoprecipitated (IP) as indicated followed by immunoblotting (IB) with streptavidin (STV). (B) RARA binds to HA-HECTD1 and ligase-deficient HA-HECTD1 C2579G (HA-HECTD11 CG ), but not to HA-NEDD4, another HECT domain-containing E3 ligase, in HEK293T cells. WCL, whole-cell lysate. (C) RARA binds HECTD1 in vivo in embryo lysates prepared from E10.5 wild-type and Hectd1 XC/XC mutant embryos. The epitope recognized by the HECTD1 antibody used for immunoprecipitation is not present in Hectd1 opm/opm mutant embryos, thus the failure of HECTD1 opm to pull down RARA serves as a negative control. (D) HECTD1 influences ubiquitination of RARA. HEK293T cells were transfected with Myc-RARA and either HA-HECTD1 or ligase-deficient HECTD1 C2579G . RARA was immunoprecipitated followed by immunoblotting with the FK2 antibody to detect poly- and monoubiquitinated RARA, or FK1 antibody to detect polyubiquitinated RARA. FK2 immunostaining indicates a slight increase in ubiquitinated RARA levels with expression of either wild-type or ligase-defective HECTD1, whereas FK1 immunostaining shows reduced ubiquitinated RARA in HA-HECTD1 C2579G transfected cells, consistent with the predicted dominant-negative activity of this construct. (E) Knockdown of HECTD1 by siRNA in HEK293T cells results in decreased ubiquitinated RARA. (F) MEFs derived from Hectd1 opm/opm and Hectd1 XC/XC mutant embryos demonstrate reduced ubiquitinated proteins in RARA immunoprecipitates and increased RARA levels in mutant compared with wild-type cells. Key positive signals are highlighted in boxes.
    Figure Legend Snippet: HECTD1 binds to RARA and influences its ubiquitination. (A) HA-RARA(281-462), binds to Myc-HECTD1(1-551) expressed in rabbit reticulocyte lysates. Proteins were immunoprecipitated (IP) as indicated followed by immunoblotting (IB) with streptavidin (STV). (B) RARA binds to HA-HECTD1 and ligase-deficient HA-HECTD1 C2579G (HA-HECTD11 CG ), but not to HA-NEDD4, another HECT domain-containing E3 ligase, in HEK293T cells. WCL, whole-cell lysate. (C) RARA binds HECTD1 in vivo in embryo lysates prepared from E10.5 wild-type and Hectd1 XC/XC mutant embryos. The epitope recognized by the HECTD1 antibody used for immunoprecipitation is not present in Hectd1 opm/opm mutant embryos, thus the failure of HECTD1 opm to pull down RARA serves as a negative control. (D) HECTD1 influences ubiquitination of RARA. HEK293T cells were transfected with Myc-RARA and either HA-HECTD1 or ligase-deficient HECTD1 C2579G . RARA was immunoprecipitated followed by immunoblotting with the FK2 antibody to detect poly- and monoubiquitinated RARA, or FK1 antibody to detect polyubiquitinated RARA. FK2 immunostaining indicates a slight increase in ubiquitinated RARA levels with expression of either wild-type or ligase-defective HECTD1, whereas FK1 immunostaining shows reduced ubiquitinated RARA in HA-HECTD1 C2579G transfected cells, consistent with the predicted dominant-negative activity of this construct. (E) Knockdown of HECTD1 by siRNA in HEK293T cells results in decreased ubiquitinated RARA. (F) MEFs derived from Hectd1 opm/opm and Hectd1 XC/XC mutant embryos demonstrate reduced ubiquitinated proteins in RARA immunoprecipitates and increased RARA levels in mutant compared with wild-type cells. Key positive signals are highlighted in boxes.

    Techniques Used: Immunoprecipitation, In Vivo, Mutagenesis, Negative Control, Transfection, Immunostaining, Expressing, Dominant Negative Mutation, Activity Assay, Construct, Derivative Assay

    11) Product Images from "Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme"

    Article Title: Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201105101

    Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, immunoprecipitation; FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.
    Figure Legend Snippet: Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, immunoprecipitation; FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.

    Techniques Used: Plasmid Preparation, Construct, Expressing, Generated, Mutagenesis, Western Blot, Activity Assay, In Vivo, Conjugation Assay, Immunoprecipitation, Quantitation Assay, Two Tailed Test

    Hectd1 physically interacts with Hsp90α. (A) Yeast two-hybrid screening of an E11.5 embryonic mouse cDNA library using Hectd1 ANK as bait detected Hsp90α (Gene ID: Hsp90aa1). Two identical clones of Hsp90α consisting of amino acids 241–459 (Hsp90αbd) of the 732–amino acid Hsp90α protein partially overlap with the first charged domain (CD1, amino acids 236–272) and the ATPase domain (amino acids 272–618) of Hsp90α. Black dots indicate locations of Hsp90 peptide fragments found by LC-MS analysis: NPDDITQEEYGEFYK (300–315), TLTIVDTGIGMTK (88–100), KADLINNLGTIAKS (100–113), and GVVDSEDLPLNISR (387–400). Peptides common to Hsp90β include: KEDQTEYLEERR (190–201), RDNSTMGYMMAKK (620–632), and YIDQEELNK (284–292). The C-terminal amino acid sequence MEEVD of Hsp90α is essential for regulated secretion. (B and C) Liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomic screening of Hectd1-binding proteins from E10.5 Hectd1 W embryo head lysates. Hectd1 was immunoprecipitated and associated proteins were resolved by 3–8% Tris-Acetate SDS-PAGE and visualized by Coomassie staining. (B) Individual bands from the Coomassie-stained gel were subjected to tryptic proteolysis, and the resulting peptides were analyzed by LC-MS/MS. (C) Representative MS/MS of a 1,293.54 D peptide. This and six other peptides (dots in A) with high XC scores (3.3–3.7) were identified as belonging to Hsp90α and Hsp90β when searched against the mouse Uniprot protein database using the Sequest algorithm as diagrammed in A. (D) Hsp90αbd binds to Hectd1 ANK in rabbit reticulocyte lysates. In vitro translated, biotinylated Hsp90αbd and Hectd1 ANK were bound and immunoprecipitated using the indicated antibodies and detected by Western blotting with streptavidin-HRP. (E and F) Hectd1 binds to Hsp90 in HEK293T cells. Cells were transfected and immunoprecipitated proteins were subjected to Western blot analyses as indicated. (E) Hsp90αbd binds to Hectd1 ANK in HEK293T cells. (F) Full-length Hsp90 and Hectd1 bind in HEK293T cells. (G) Hsp90 binds to Hectd1 in the developing embryo. Hectd1 was immunoprecipitated from lysates prepared from E12.5 Hectd1 W and Hectd1 O embryo heads and subjected to Western blot analysis as indicated ( n = 4). (H and I) Hsp90 binds to Hectd1 but not the related HECT domain containing Nedd4 Ub ligase. (H and I) HEK293T cells were transfected with HA-Nedd4 or HA-Hectd1 along with Myc-Hsp90. They were then HA immunoprocipitated and subjected to Western blotting (H), or Hsp90 (I) was immunoprecipitated from E12.5 Hectd1 W and Hectd1 O embryo head lysates followed by Western blotting to detect Nedd4 and Hectd1. All data are representative of three independent experiments unless otherwise indicated. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation.
    Figure Legend Snippet: Hectd1 physically interacts with Hsp90α. (A) Yeast two-hybrid screening of an E11.5 embryonic mouse cDNA library using Hectd1 ANK as bait detected Hsp90α (Gene ID: Hsp90aa1). Two identical clones of Hsp90α consisting of amino acids 241–459 (Hsp90αbd) of the 732–amino acid Hsp90α protein partially overlap with the first charged domain (CD1, amino acids 236–272) and the ATPase domain (amino acids 272–618) of Hsp90α. Black dots indicate locations of Hsp90 peptide fragments found by LC-MS analysis: NPDDITQEEYGEFYK (300–315), TLTIVDTGIGMTK (88–100), KADLINNLGTIAKS (100–113), and GVVDSEDLPLNISR (387–400). Peptides common to Hsp90β include: KEDQTEYLEERR (190–201), RDNSTMGYMMAKK (620–632), and YIDQEELNK (284–292). The C-terminal amino acid sequence MEEVD of Hsp90α is essential for regulated secretion. (B and C) Liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomic screening of Hectd1-binding proteins from E10.5 Hectd1 W embryo head lysates. Hectd1 was immunoprecipitated and associated proteins were resolved by 3–8% Tris-Acetate SDS-PAGE and visualized by Coomassie staining. (B) Individual bands from the Coomassie-stained gel were subjected to tryptic proteolysis, and the resulting peptides were analyzed by LC-MS/MS. (C) Representative MS/MS of a 1,293.54 D peptide. This and six other peptides (dots in A) with high XC scores (3.3–3.7) were identified as belonging to Hsp90α and Hsp90β when searched against the mouse Uniprot protein database using the Sequest algorithm as diagrammed in A. (D) Hsp90αbd binds to Hectd1 ANK in rabbit reticulocyte lysates. In vitro translated, biotinylated Hsp90αbd and Hectd1 ANK were bound and immunoprecipitated using the indicated antibodies and detected by Western blotting with streptavidin-HRP. (E and F) Hectd1 binds to Hsp90 in HEK293T cells. Cells were transfected and immunoprecipitated proteins were subjected to Western blot analyses as indicated. (E) Hsp90αbd binds to Hectd1 ANK in HEK293T cells. (F) Full-length Hsp90 and Hectd1 bind in HEK293T cells. (G) Hsp90 binds to Hectd1 in the developing embryo. Hectd1 was immunoprecipitated from lysates prepared from E12.5 Hectd1 W and Hectd1 O embryo heads and subjected to Western blot analysis as indicated ( n = 4). (H and I) Hsp90 binds to Hectd1 but not the related HECT domain containing Nedd4 Ub ligase. (H and I) HEK293T cells were transfected with HA-Nedd4 or HA-Hectd1 along with Myc-Hsp90. They were then HA immunoprocipitated and subjected to Western blotting (H), or Hsp90 (I) was immunoprecipitated from E12.5 Hectd1 W and Hectd1 O embryo head lysates followed by Western blotting to detect Nedd4 and Hectd1. All data are representative of three independent experiments unless otherwise indicated. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation.

    Techniques Used: Two Hybrid Screening, cDNA Library Assay, Clone Assay, Liquid Chromatography with Mass Spectroscopy, Sequencing, Liquid Chromatography, Mass Spectrometry, Binding Assay, Immunoprecipitation, SDS Page, Staining, In Vitro, Western Blot, Transfection

    Hectd1 is required for K63-linked Ub n of Hsp90. (A–D) HEK293T cells were transfected, and lysates were subjected to immunoprecipitation and Western blot analysis as indicated. (A) Ubiquitination of Myc-Hsp90 increases with expression of HA-Hectd1 ( n = 2). (B) siRNA-mediated knockdown of endogenous Hectd1 reduces the accumulation of HMW-Hsp90α ( n = 2). (C) Hsp90α ubiquitination utilizes K63 linkages. (D) Hectd1-dependent polyubiquitination of Hsp90 occurs primarily through K63 linkages. (E) HMW Hsp90 species are reduced in Hectd1 mutant heads. E12.5 Hectd1 W (W) and Hectd1 X (X) embryos were cultured in the presence of 10 µM MG132 for 3 h before lysis and immunoprecipitation of Hectd1. Immunoprecipitates were subjected to Western blot analyses to detect Hsp90 that coimmunoprecipitated with Hectd1. (F) Hsp90 ubiquitination is reduced in CM cultures from Hectd1 O (O) and Hectd1 X (X) mutants compared with Hectd1 W (W). Hsp90 was immunoprecipitated from E12.5 CM primary cultures in highly denaturing ubiquitination buffer plus 5% SDS and subjected to Western blot analyses as indicated. The appearance of a 30-kD ubiquitinated protein (asterisk) is reduced in Hectd1 mutant cells. All data are representative of three independent experiments unless otherwise indicated. Abbreviations: W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation; (Ub)n, Ub n ; wt-Ub, wild-type Ub; K48R, mutant Ub lysine 48 arginine; K63R, mutant Ub lysine 48 arginine; K0, lysineless Ub.
    Figure Legend Snippet: Hectd1 is required for K63-linked Ub n of Hsp90. (A–D) HEK293T cells were transfected, and lysates were subjected to immunoprecipitation and Western blot analysis as indicated. (A) Ubiquitination of Myc-Hsp90 increases with expression of HA-Hectd1 ( n = 2). (B) siRNA-mediated knockdown of endogenous Hectd1 reduces the accumulation of HMW-Hsp90α ( n = 2). (C) Hsp90α ubiquitination utilizes K63 linkages. (D) Hectd1-dependent polyubiquitination of Hsp90 occurs primarily through K63 linkages. (E) HMW Hsp90 species are reduced in Hectd1 mutant heads. E12.5 Hectd1 W (W) and Hectd1 X (X) embryos were cultured in the presence of 10 µM MG132 for 3 h before lysis and immunoprecipitation of Hectd1. Immunoprecipitates were subjected to Western blot analyses to detect Hsp90 that coimmunoprecipitated with Hectd1. (F) Hsp90 ubiquitination is reduced in CM cultures from Hectd1 O (O) and Hectd1 X (X) mutants compared with Hectd1 W (W). Hsp90 was immunoprecipitated from E12.5 CM primary cultures in highly denaturing ubiquitination buffer plus 5% SDS and subjected to Western blot analyses as indicated. The appearance of a 30-kD ubiquitinated protein (asterisk) is reduced in Hectd1 mutant cells. All data are representative of three independent experiments unless otherwise indicated. Abbreviations: W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation; (Ub)n, Ub n ; wt-Ub, wild-type Ub; K48R, mutant Ub lysine 48 arginine; K63R, mutant Ub lysine 48 arginine; K0, lysineless Ub.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Expressing, Mutagenesis, Cell Culture, Lysis

    12) Product Images from "NRF2 regulates serine biosynthesis in non-small cell lung cancer"

    Article Title: NRF2 regulates serine biosynthesis in non-small cell lung cancer

    Journal: Nature genetics

    doi: 10.1038/ng.3421

    NRF2 regulates the expression of serine/glycine biosynthesis genes through ATF4. (a) ATF4 mRNA expression in A549 cells expressing scramble shRNA (SCR), or NRF2 shRNA #1. (b) Western blot of NRF2, ATF4 and ACTIN expression in cells from (a). (c) Western blot of NRF2, ATF4 and serine pathway enzyme expression in lysates from A549s expressing scramble (SCR), NRF2 shRNA #1, or ATF4 shRNAs #1 or #2. (d) mRNA expression in cells from (c). (e) ATF4 knockdown impairs serine biosynthesis. Cell lines from were grown in the presence of U- 13 C-glucose for the indicated time points, the metabolites extracted and the fractional 13 C-labeling on serine analysed by LC/MS. (f) ATF4 rescues serine biosynthesis enzyme expression following NRF2 knockdown. A549 cells were infected with lentivirus encoding mATF4 prior to infection with scramble or NRF2-targeting lentivirus. (g) Western analysis of NRF2, ATF4, and ACTIN expression in the cells from (f). (h) ATF4 rescues the serine biosynthesis defect in shNRF2 A549 cells. Cells were assayed as in (e). (i) ATF4 rescues the growth of H1975 cells in serine deficient media. Cells expressing luciferase (LUC) or ATF4 were grown in the indicated media for 3 days and cell number normalized to cells grown in full media. (j) Chromatin immunoprecipitation of ATF4 to the PHGDH, PSAT1 and SHMT2 promoters. Samples were normalized to IgG control immunoprecipitations. Results are the average of 3 technical (a, d, f, j) or biological (e, h, i) replicates.
    Figure Legend Snippet: NRF2 regulates the expression of serine/glycine biosynthesis genes through ATF4. (a) ATF4 mRNA expression in A549 cells expressing scramble shRNA (SCR), or NRF2 shRNA #1. (b) Western blot of NRF2, ATF4 and ACTIN expression in cells from (a). (c) Western blot of NRF2, ATF4 and serine pathway enzyme expression in lysates from A549s expressing scramble (SCR), NRF2 shRNA #1, or ATF4 shRNAs #1 or #2. (d) mRNA expression in cells from (c). (e) ATF4 knockdown impairs serine biosynthesis. Cell lines from were grown in the presence of U- 13 C-glucose for the indicated time points, the metabolites extracted and the fractional 13 C-labeling on serine analysed by LC/MS. (f) ATF4 rescues serine biosynthesis enzyme expression following NRF2 knockdown. A549 cells were infected with lentivirus encoding mATF4 prior to infection with scramble or NRF2-targeting lentivirus. (g) Western analysis of NRF2, ATF4, and ACTIN expression in the cells from (f). (h) ATF4 rescues the serine biosynthesis defect in shNRF2 A549 cells. Cells were assayed as in (e). (i) ATF4 rescues the growth of H1975 cells in serine deficient media. Cells expressing luciferase (LUC) or ATF4 were grown in the indicated media for 3 days and cell number normalized to cells grown in full media. (j) Chromatin immunoprecipitation of ATF4 to the PHGDH, PSAT1 and SHMT2 promoters. Samples were normalized to IgG control immunoprecipitations. Results are the average of 3 technical (a, d, f, j) or biological (e, h, i) replicates.

    Techniques Used: Expressing, shRNA, Western Blot, Labeling, Liquid Chromatography with Mass Spectroscopy, Infection, Luciferase, Chromatin Immunoprecipitation

    13) Product Images from "Tamm-Horsfall Protein Regulates Circulating and Renal Cytokines by Affecting Glomerular Filtration Rate and Acting as a Urinary Cytokine Trap *"

    Article Title: Tamm-Horsfall Protein Regulates Circulating and Renal Cytokines by Affecting Glomerular Filtration Rate and Acting as a Urinary Cytokine Trap *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.348243

    In vivo binding of renal cytokines to THP. Top panels , co-immunoprecipitation of THP-bound cytokines followed by cytokine ELISA. Total kidney protein extracts from wild-type ( WT ) and THP KO mice (2 representative mice per genotype shown) were subject to IP using a rabbit anti-THP antibody. The IP products were loaded into microtiter wells pre-coated with antibodies against IFN-γ ( A ), IL1α ( B ), TNF-α ( C ), or IL13 ( D ), to capture THP-bound cytokines. This was followed by conventional ELISA (see “Experimental Procedures” for details). Starting materials for IP contained the same amount of total kidney proteins. Lower panels , ELISA quantification of renal cytokine input ( e.g. THP-bound and -unbound cytokines), expressed as per milligram of total kidney proteins. Note that, whereas THP KO mice had higher renal cytokines ( lower panels , filled bars ), little could be precipitated with anti-THP antibody ( upper panels , filled bars ). In contrast, in wild-type mice, each of the four tested cytokines could be precipitated by the anti-THP antibody ( upper panels , open bars ) strongly suggesting an in vivo THP-cytokine interaction (see text for details).
    Figure Legend Snippet: In vivo binding of renal cytokines to THP. Top panels , co-immunoprecipitation of THP-bound cytokines followed by cytokine ELISA. Total kidney protein extracts from wild-type ( WT ) and THP KO mice (2 representative mice per genotype shown) were subject to IP using a rabbit anti-THP antibody. The IP products were loaded into microtiter wells pre-coated with antibodies against IFN-γ ( A ), IL1α ( B ), TNF-α ( C ), or IL13 ( D ), to capture THP-bound cytokines. This was followed by conventional ELISA (see “Experimental Procedures” for details). Starting materials for IP contained the same amount of total kidney proteins. Lower panels , ELISA quantification of renal cytokine input ( e.g. THP-bound and -unbound cytokines), expressed as per milligram of total kidney proteins. Note that, whereas THP KO mice had higher renal cytokines ( lower panels , filled bars ), little could be precipitated with anti-THP antibody ( upper panels , filled bars ). In contrast, in wild-type mice, each of the four tested cytokines could be precipitated by the anti-THP antibody ( upper panels , open bars ) strongly suggesting an in vivo THP-cytokine interaction (see text for details).

    Techniques Used: In Vivo, Binding Assay, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Mouse Assay

    14) Product Images from "The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch"

    Article Title: The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.036491

    HECTD1 binds to RARA and influences its ubiquitination. (A) HA-RARA(281-462), binds to Myc-HECTD1(1-551) expressed in rabbit reticulocyte lysates. Proteins were immunoprecipitated (IP) as indicated followed by immunoblotting (IB) with streptavidin (STV). (B) RARA binds to HA-HECTD1 and ligase-deficient HA-HECTD1 C2579G (HA-HECTD11 CG ), but not to HA-NEDD4, another HECT domain-containing E3 ligase, in HEK293T cells. WCL, whole-cell lysate. (C) RARA binds HECTD1 in vivo in embryo lysates prepared from E10.5 wild-type and Hectd1 XC/XC mutant embryos. The epitope recognized by the HECTD1 antibody used for immunoprecipitation is not present in Hectd1 opm/opm mutant embryos, thus the failure of HECTD1 opm to pull down RARA serves as a negative control. (D) HECTD1 influences ubiquitination of RARA. HEK293T cells were transfected with Myc-RARA and either HA-HECTD1 or ligase-deficient HECTD1 C2579G . RARA was immunoprecipitated followed by immunoblotting with the FK2 antibody to detect poly- and monoubiquitinated RARA, or FK1 antibody to detect polyubiquitinated RARA. FK2 immunostaining indicates a slight increase in ubiquitinated RARA levels with expression of either wild-type or ligase-defective HECTD1, whereas FK1 immunostaining shows reduced ubiquitinated RARA in HA-HECTD1 C2579G transfected cells, consistent with the predicted dominant-negative activity of this construct. (E) Knockdown of HECTD1 by siRNA in HEK293T cells results in decreased ubiquitinated RARA. (F) MEFs derived from Hectd1 opm/opm and Hectd1 XC/XC mutant embryos demonstrate reduced ubiquitinated proteins in RARA immunoprecipitates and increased RARA levels in mutant compared with wild-type cells. Key positive signals are highlighted in boxes.
    Figure Legend Snippet: HECTD1 binds to RARA and influences its ubiquitination. (A) HA-RARA(281-462), binds to Myc-HECTD1(1-551) expressed in rabbit reticulocyte lysates. Proteins were immunoprecipitated (IP) as indicated followed by immunoblotting (IB) with streptavidin (STV). (B) RARA binds to HA-HECTD1 and ligase-deficient HA-HECTD1 C2579G (HA-HECTD11 CG ), but not to HA-NEDD4, another HECT domain-containing E3 ligase, in HEK293T cells. WCL, whole-cell lysate. (C) RARA binds HECTD1 in vivo in embryo lysates prepared from E10.5 wild-type and Hectd1 XC/XC mutant embryos. The epitope recognized by the HECTD1 antibody used for immunoprecipitation is not present in Hectd1 opm/opm mutant embryos, thus the failure of HECTD1 opm to pull down RARA serves as a negative control. (D) HECTD1 influences ubiquitination of RARA. HEK293T cells were transfected with Myc-RARA and either HA-HECTD1 or ligase-deficient HECTD1 C2579G . RARA was immunoprecipitated followed by immunoblotting with the FK2 antibody to detect poly- and monoubiquitinated RARA, or FK1 antibody to detect polyubiquitinated RARA. FK2 immunostaining indicates a slight increase in ubiquitinated RARA levels with expression of either wild-type or ligase-defective HECTD1, whereas FK1 immunostaining shows reduced ubiquitinated RARA in HA-HECTD1 C2579G transfected cells, consistent with the predicted dominant-negative activity of this construct. (E) Knockdown of HECTD1 by siRNA in HEK293T cells results in decreased ubiquitinated RARA. (F) MEFs derived from Hectd1 opm/opm and Hectd1 XC/XC mutant embryos demonstrate reduced ubiquitinated proteins in RARA immunoprecipitates and increased RARA levels in mutant compared with wild-type cells. Key positive signals are highlighted in boxes.

    Techniques Used: Immunoprecipitation, In Vivo, Mutagenesis, Negative Control, Transfection, Immunostaining, Expressing, Dominant Negative Mutation, Activity Assay, Construct, Derivative Assay

    15) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.046649

    Hhex and SOX13 competitive immunoprecipitation assay. A lysate from 293T cells co-transfected with SOX13 -FLAG- and TCF1 -myc-tagged-expressing plasmids was immunoprecipitated ( IP ) using anti-FLAG antibody in the absence of GST proteins ( lanes 1 and 4 )
    Figure Legend Snippet: Hhex and SOX13 competitive immunoprecipitation assay. A lysate from 293T cells co-transfected with SOX13 -FLAG- and TCF1 -myc-tagged-expressing plasmids was immunoprecipitated ( IP ) using anti-FLAG antibody in the absence of GST proteins ( lanes 1 and 4 )

    Techniques Used: Immunoprecipitation, Transfection, Expressing

    16) Product Images from "Short RNA Molecules with High Binding Affinity to the KH Motif of A-Kinase Anchoring Protein 1 (AKAP1): Implications for the Regulation of Steroidogenesis"

    Article Title: Short RNA Molecules with High Binding Affinity to the KH Motif of A-Kinase Anchoring Protein 1 (AKAP1): Implications for the Regulation of Steroidogenesis

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2012-1123

    STAR mRNA associates with AKAP1 in vivo in the human adrenocortical carcinoma cell line H295R as determined by UV cross-linking, followed by immunoprecipitation and RT-PCR. A, Representative Western blot with antibodies against AKAP1 or β-actin
    Figure Legend Snippet: STAR mRNA associates with AKAP1 in vivo in the human adrenocortical carcinoma cell line H295R as determined by UV cross-linking, followed by immunoprecipitation and RT-PCR. A, Representative Western blot with antibodies against AKAP1 or β-actin

    Techniques Used: In Vivo, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Western Blot

    17) Product Images from "Endocytic Recycling Proteins EHD1 and EHD2 Interact with Fer-1-like-5 (Fer1L5) and Mediate Myoblast Fusion *"

    Article Title: Endocytic Recycling Proteins EHD1 and EHD2 Interact with Fer-1-like-5 (Fer1L5) and Mediate Myoblast Fusion *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.157222

    A , all six Fer1L5 C2 domains were fused to glutathione S -transferase, transferred to polyvinylidene difluoride membrane and, in this experiment, incubated with 35 S-labeled EHD1. EHD1 bound to the C2B domain of Fer1L5 and had a smaller interaction with the C2E and C2F domains. Nonspecific bacterial proteins can be identified at 36 and 32 kDa. B , an anti-Fer1L5 antibody was used to immunoprecipitate ( IP ) C2C12 lysates, and immunoblotting ( IB ) with an anti-EHD1 antibody demonstrated that EHD1 associates with Fer1L5. The Input lane contains 25 μg of cellular lysate, which does not contain enough Fer1L5 for immunoreactivity. Immunoprecipitation used 200 μg of cellular lysate. C , mutation of the NPF motif within the C2B domain to SPL or CPF decreased but did not eliminate binding of EHD1. Ctrl , control.
    Figure Legend Snippet: A , all six Fer1L5 C2 domains were fused to glutathione S -transferase, transferred to polyvinylidene difluoride membrane and, in this experiment, incubated with 35 S-labeled EHD1. EHD1 bound to the C2B domain of Fer1L5 and had a smaller interaction with the C2E and C2F domains. Nonspecific bacterial proteins can be identified at 36 and 32 kDa. B , an anti-Fer1L5 antibody was used to immunoprecipitate ( IP ) C2C12 lysates, and immunoblotting ( IB ) with an anti-EHD1 antibody demonstrated that EHD1 associates with Fer1L5. The Input lane contains 25 μg of cellular lysate, which does not contain enough Fer1L5 for immunoreactivity. Immunoprecipitation used 200 μg of cellular lysate. C , mutation of the NPF motif within the C2B domain to SPL or CPF decreased but did not eliminate binding of EHD1. Ctrl , control.

    Techniques Used: Incubation, Labeling, Immunoprecipitation, Mutagenesis, Binding Assay

    A , a blot overlay shows the direct interaction between EHD2 and Fer1L5. All six Fer1L5 C2 domains were expressed in bacteria as fusion proteins with glutathione S -transferase, transferred to polyvinylidene difluoride membrane, and incubated with 35 S-labeled EHD2. The upper panel shows the Coomassie-stained blots of GST-C2 fusion proteins. The lower panel shows the results after binding to radiolabeled EHD2. EHD2 strongly interacted with the C2B domain of Fer1L5. Less interaction was detected between EHD2 and the Fer1L5 C2E domain. Nonspecific bacterial proteins can be identified at 32kDa. B , an anti-Fer1L5 antibody was used to immunoprecipitate ( IP ) lysates from differentiated C2C12 cells, and these lysates were then immunoblotted ( IB ) with an anti-EHD2 and anti-Fer1L5 antibody. Immunoprecipitation with anti-Fer1L5 resulted in the precipitation with EHD2. The Input lane contains 25 μg of cellular lysate, which does not contain enough Fer1L5 for immunoreactivity. Immunoprecipitation used 200 μg of cellular lysate. C , mutation of the NPF motif within the C2B domain to SPL or CPF decreased, but did not abolish, binding of EHD2. Ctrl , control.
    Figure Legend Snippet: A , a blot overlay shows the direct interaction between EHD2 and Fer1L5. All six Fer1L5 C2 domains were expressed in bacteria as fusion proteins with glutathione S -transferase, transferred to polyvinylidene difluoride membrane, and incubated with 35 S-labeled EHD2. The upper panel shows the Coomassie-stained blots of GST-C2 fusion proteins. The lower panel shows the results after binding to radiolabeled EHD2. EHD2 strongly interacted with the C2B domain of Fer1L5. Less interaction was detected between EHD2 and the Fer1L5 C2E domain. Nonspecific bacterial proteins can be identified at 32kDa. B , an anti-Fer1L5 antibody was used to immunoprecipitate ( IP ) lysates from differentiated C2C12 cells, and these lysates were then immunoblotted ( IB ) with an anti-EHD2 and anti-Fer1L5 antibody. Immunoprecipitation with anti-Fer1L5 resulted in the precipitation with EHD2. The Input lane contains 25 μg of cellular lysate, which does not contain enough Fer1L5 for immunoreactivity. Immunoprecipitation used 200 μg of cellular lysate. C , mutation of the NPF motif within the C2B domain to SPL or CPF decreased, but did not abolish, binding of EHD2. Ctrl , control.

    Techniques Used: Incubation, Labeling, Staining, Binding Assay, Immunoprecipitation, Mutagenesis

    18) Product Images from "Peptide Lv augments L-type voltage-gated calcium channels through vascular endothelial growth factor receptor 2 (VEGFR2) signaling"

    Article Title: Peptide Lv augments L-type voltage-gated calcium channels through vascular endothelial growth factor receptor 2 (VEGFR2) signaling

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamcr.2015.02.007

    VEGFR2 interacts with peptide Lv (A) Mouse whole brain lysate samples were incubated with the anti-peptide Lv antibody conjugated with sepharose 4B to determine potential receptors for peptide Lv. Four major bands indicated by arrows with molecular weights ranging from 40 kD to 200 kD were excised for MALDI-TOF analyses. (B, C) Verification of the interaction between peptide Lv and VEGFR2 was carried out in chicken embryonic heart lysate using co-immunoprecipitation (co-IP). (B) Anti-peptide Lv antibody was able to pull down VEGFR2(*). (C) Likewise, anti-VEGFR2 antibody was able to pull down peptide Lv (#). The rabbit IgG (Rabbit Ig) was used as a control.
    Figure Legend Snippet: VEGFR2 interacts with peptide Lv (A) Mouse whole brain lysate samples were incubated with the anti-peptide Lv antibody conjugated with sepharose 4B to determine potential receptors for peptide Lv. Four major bands indicated by arrows with molecular weights ranging from 40 kD to 200 kD were excised for MALDI-TOF analyses. (B, C) Verification of the interaction between peptide Lv and VEGFR2 was carried out in chicken embryonic heart lysate using co-immunoprecipitation (co-IP). (B) Anti-peptide Lv antibody was able to pull down VEGFR2(*). (C) Likewise, anti-VEGFR2 antibody was able to pull down peptide Lv (#). The rabbit IgG (Rabbit Ig) was used as a control.

    Techniques Used: Incubation, Immunoprecipitation, Co-Immunoprecipitation Assay

    19) Product Images from "Intracellular Shuttling and Mitochondrial Function of Thioredoxin-interacting Protein *"

    Article Title: Intracellular Shuttling and Mitochondrial Function of Thioredoxin-interacting Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.034421

    TXNIP interaction with mitochondrial Trx2. A , effects of oxidative stress on TXNIP-Trx2 co-immunoprecipitation. INS-1 cells were treated without (control ( C )) or with H 2 O 2 (15 μ m for 4 h) prior to isolation of their mitochondrial fractions and
    Figure Legend Snippet: TXNIP interaction with mitochondrial Trx2. A , effects of oxidative stress on TXNIP-Trx2 co-immunoprecipitation. INS-1 cells were treated without (control ( C )) or with H 2 O 2 (15 μ m for 4 h) prior to isolation of their mitochondrial fractions and

    Techniques Used: Immunoprecipitation, Isolation

    20) Product Images from "Cellular distribution of the IGF-1R in corneal epithelial cells"

    Article Title: Cellular distribution of the IGF-1R in corneal epithelial cells

    Journal: Experimental Eye Research

    doi: 10.1016/j.exer.2011.12.006

    IGF-1R:E-cadherin complex. Co-immunoprecipitation of hTCEpi whole cell lysates using two separate IGF-1R antibodies confirmed an association between IGF-1Rβ and E-cadherin. (A) IGF-1Rβ was immunoprecipitated using a rabbit polyclonal antibody (Cell Signaling). (B) IGF-1Rβ clone C-20 was immunoprecipitated using a rabbit polyclonal antibody (Santa Cruz). Blotting for IGF-1Rβ was used to confirm successful immunoprecipitation. An irrelevant IgG was used as a control.
    Figure Legend Snippet: IGF-1R:E-cadherin complex. Co-immunoprecipitation of hTCEpi whole cell lysates using two separate IGF-1R antibodies confirmed an association between IGF-1Rβ and E-cadherin. (A) IGF-1Rβ was immunoprecipitated using a rabbit polyclonal antibody (Cell Signaling). (B) IGF-1Rβ clone C-20 was immunoprecipitated using a rabbit polyclonal antibody (Santa Cruz). Blotting for IGF-1Rβ was used to confirm successful immunoprecipitation. An irrelevant IgG was used as a control.

    Techniques Used: Immunoprecipitation

    21) Product Images from "Modulation of Vitamin D Receptor Activity by the Corepressor Hairless: Differential Effects of Hairless Isoforms"

    Article Title: Modulation of Vitamin D Receptor Activity by the Corepressor Hairless: Differential Effects of Hairless Isoforms

    Journal: Endocrinology

    doi: 10.1210/en.2009-0358

    Interactions of the HR isoforms with VDR and HDAC1. A, The HR isoforms interact with VDR. VDR was coexpressed with Flag-tagged HRs in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-VDR antibody (WB:VDR). B, Interactions of HDAC1 with the HR isoforms. HA-tagged HDAC1 was coexpressed with Flag-tagged HRs in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-HA antibody (WB:HA). In, Input; IP, immunoprecipitation; NS, nonspecific.
    Figure Legend Snippet: Interactions of the HR isoforms with VDR and HDAC1. A, The HR isoforms interact with VDR. VDR was coexpressed with Flag-tagged HRs in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-VDR antibody (WB:VDR). B, Interactions of HDAC1 with the HR isoforms. HA-tagged HDAC1 was coexpressed with Flag-tagged HRs in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-HA antibody (WB:HA). In, Input; IP, immunoprecipitation; NS, nonspecific.

    Techniques Used: Immunoprecipitation, Western Blot

    Mutations in 55-amino acid region in the full-length HR disrupt HDAC1 binding. A, COS-7 cells were transfected with pSG5 and p3XFLAG-CMV-7.1 vectors (control), pSG5-VDR and p3XFLAG–CMV-7.1 expression vectors (VDR), pSG5-VDR and p3XFLAG-HRE1100A/E1101A-CMV-7.1 (HR2E2A), and the 24-hydroxylase promoter luciferase reporter. Cells were treated with graded concentrations of 1,25(OH) 2 D 3 for 24 h and then assayed for luciferase activity. The E1100A/E1101A double mutation in the HR cDNA abolishes corepressor activity and disrupts interaction with HDAC1. Inset is immunoblot of HR E1100A/E1101A protein (HR2E2A). B, HA-tagged HDAC1 was coexpressed with Flag-tagged full-length HR or HR E1100A/E1101A in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-HA antibody (WB:HA). In, Input; IP, immunoprecipitation; RLU, relative light units.
    Figure Legend Snippet: Mutations in 55-amino acid region in the full-length HR disrupt HDAC1 binding. A, COS-7 cells were transfected with pSG5 and p3XFLAG-CMV-7.1 vectors (control), pSG5-VDR and p3XFLAG–CMV-7.1 expression vectors (VDR), pSG5-VDR and p3XFLAG-HRE1100A/E1101A-CMV-7.1 (HR2E2A), and the 24-hydroxylase promoter luciferase reporter. Cells were treated with graded concentrations of 1,25(OH) 2 D 3 for 24 h and then assayed for luciferase activity. The E1100A/E1101A double mutation in the HR cDNA abolishes corepressor activity and disrupts interaction with HDAC1. Inset is immunoblot of HR E1100A/E1101A protein (HR2E2A). B, HA-tagged HDAC1 was coexpressed with Flag-tagged full-length HR or HR E1100A/E1101A in COS-7 cells. Proteins were immunoprecipitated with anti-Flag antibody (IP:Flag) and detected by immunoblot with anti-HA antibody (WB:HA). In, Input; IP, immunoprecipitation; RLU, relative light units.

    Techniques Used: Binding Assay, Transfection, Expressing, Luciferase, Activity Assay, Mutagenesis, Immunoprecipitation, Western Blot

    22) Product Images from "Analysis of Proteolytic Processes and Enzymatic Activities in the Generation of Huntingtin N-Terminal Fragments in an HEK293 Cell Model"

    Article Title: Analysis of Proteolytic Processes and Enzymatic Activities in the Generation of Huntingtin N-Terminal Fragments in an HEK293 Cell Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0050750

    Htt cleavage occurs C-terminal to cysteine 105. A, The sequence of htt is shown through residue 171, the substrate used in these experiments. The first cysteine in htt is at residue 105 (bold) and represents the most N-terminal cysteine that could be labeled with sulfhydrylated biotin (Biotin-SH). This cysteine must be present for any labeling to occur. Subsequent immunoprecipitation (IP) followed by detection with streptavidin or htt antibodies was used to visualize the biotinylation. B, Detection using streptavidin-HRP of transfected cell lysate (Tfx N171-18Q, +) reacted with biotinylation reagent (Biotin, +) shows cp-B/2 and cp-A/1-sized products (arrows). Asterisks (*) signify possible multimers of htt. C, Detection of htt with the antibody htt3-16 confirms the identities of cp-B/2 and cp-A/1 (bottom two arrows). IgG from the immunoprecipitation is observed in all lanes (top arrow). The images shown are representative of at least 3 repetitions of the experiment.
    Figure Legend Snippet: Htt cleavage occurs C-terminal to cysteine 105. A, The sequence of htt is shown through residue 171, the substrate used in these experiments. The first cysteine in htt is at residue 105 (bold) and represents the most N-terminal cysteine that could be labeled with sulfhydrylated biotin (Biotin-SH). This cysteine must be present for any labeling to occur. Subsequent immunoprecipitation (IP) followed by detection with streptavidin or htt antibodies was used to visualize the biotinylation. B, Detection using streptavidin-HRP of transfected cell lysate (Tfx N171-18Q, +) reacted with biotinylation reagent (Biotin, +) shows cp-B/2 and cp-A/1-sized products (arrows). Asterisks (*) signify possible multimers of htt. C, Detection of htt with the antibody htt3-16 confirms the identities of cp-B/2 and cp-A/1 (bottom two arrows). IgG from the immunoprecipitation is observed in all lanes (top arrow). The images shown are representative of at least 3 repetitions of the experiment.

    Techniques Used: Sequencing, Labeling, Immunoprecipitation, Transfection

    23) Product Images from "TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle"

    Article Title: TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle

    Journal: Nature

    doi: 10.1038/s41586-018-0665-2

    TDP-43 adopts higher-ordered state during normal skeletal muscle formation (Related to Fig 1 ). (a) Secondary antibody control for 5 dpi TDP-43 staining in TA muscle sections. (n = 5 mice per condition providing similar results). eMHC (embryonic myosin heavy chain) immunoreactivity in regenerating myofibers with nuclei counterstained with DAPI. (b) Immunoreactive TDP-43 in 30dpi TA muscle sections with nuclei counterstained with DAPI (n = 4 mice). Scale bar is 50 μm. (c) RIPA-UREA assay reveals presence of a urea insoluble TDP-43 fraction isolated from C2C12 myotubes differentiated for 7 days but not in C2C12 myoblasts (n=3 independent experiments, each giving similar results, unpaired two-tailed t-test, p-value = 0.0008). GAPDH remains RIPA-soluble in both myoblasts and myotubes (n = 3 independent experiments, each giving similar results, unpaired two-tailed t-test, p-value = 0.7443) (d) Higher molecular weight SDS-resistant TDP-43 assemblies present in differentiating C2C12 myotubes resolved by SDD-AGE (Semi-Denaturing Detergent Agarose Gel Electrophoresis) (n = 3 independent experiments). Pub1 Q/N-GFP from yeast forms higher molecular weight SDS-resistant assemblies than TDP-43 assemblies. (e) Schematic for the isolation of myo-granules containing TDP-43 during skeletal muscle formation (f) Immunoprecipitation (IP) of TDP-43 on dynabeads (DB) reveals oligomers isolated from C2C12 myotubes but absent in myoblasts as observed by electron microscopy (EM) (n = 3 independent experiments). (g) Stress granule formation in multinucleated myotubes derived from C2C12 cells. Immunofluorescence using antibodies agains t stress granule proteins, G3BP1 and Pabp1, after ± NaAsO 2 treatment for 60min (n=3 independent experiments, each giving similar results ) . Zoom represents magnified inset. Scale bars are 5μm and 20μm, respectively.
    Figure Legend Snippet: TDP-43 adopts higher-ordered state during normal skeletal muscle formation (Related to Fig 1 ). (a) Secondary antibody control for 5 dpi TDP-43 staining in TA muscle sections. (n = 5 mice per condition providing similar results). eMHC (embryonic myosin heavy chain) immunoreactivity in regenerating myofibers with nuclei counterstained with DAPI. (b) Immunoreactive TDP-43 in 30dpi TA muscle sections with nuclei counterstained with DAPI (n = 4 mice). Scale bar is 50 μm. (c) RIPA-UREA assay reveals presence of a urea insoluble TDP-43 fraction isolated from C2C12 myotubes differentiated for 7 days but not in C2C12 myoblasts (n=3 independent experiments, each giving similar results, unpaired two-tailed t-test, p-value = 0.0008). GAPDH remains RIPA-soluble in both myoblasts and myotubes (n = 3 independent experiments, each giving similar results, unpaired two-tailed t-test, p-value = 0.7443) (d) Higher molecular weight SDS-resistant TDP-43 assemblies present in differentiating C2C12 myotubes resolved by SDD-AGE (Semi-Denaturing Detergent Agarose Gel Electrophoresis) (n = 3 independent experiments). Pub1 Q/N-GFP from yeast forms higher molecular weight SDS-resistant assemblies than TDP-43 assemblies. (e) Schematic for the isolation of myo-granules containing TDP-43 during skeletal muscle formation (f) Immunoprecipitation (IP) of TDP-43 on dynabeads (DB) reveals oligomers isolated from C2C12 myotubes but absent in myoblasts as observed by electron microscopy (EM) (n = 3 independent experiments). (g) Stress granule formation in multinucleated myotubes derived from C2C12 cells. Immunofluorescence using antibodies agains t stress granule proteins, G3BP1 and Pabp1, after ± NaAsO 2 treatment for 60min (n=3 independent experiments, each giving similar results ) . Zoom represents magnified inset. Scale bars are 5μm and 20μm, respectively.

    Techniques Used: Staining, Mouse Assay, Isolation, Two Tailed Test, Molecular Weight, Agarose Gel Electrophoresis, Immunoprecipitation, Electron Microscopy, Derivative Assay, Immunofluorescence

    Myo-granules containing TDP-43 are amyloid-like oligomers. (Related to Fig 2 ). (a-b) X-ray diffraction on immunoprecipitated myo-granules (right half of both panels) compared to the diffraction of mock IgG immunoprecipitation (left half of panel a) and to the diffraction of super oxide dismutase 1 (SOD1) amyloid oligomers (left half of panel b). In all diffraction patterns two rings at ~4.8 Å and ~10 Å are drawn on the bottom half to highlight absence of an ~4.8 Å reflection in the mock immunoprecipitation and a similar ~4.8 Å reflection with a ~10 Å reflection absence in the SOD1 diffraction. One sample per condition was used. Two diffraction images at different rotations were taken per sample and each image gave similar results. (c) Complexes immunopurified using TDP-43 or (d) A11 isolated from C2C12 myotubes are immunoreactive for A11 and TDP-43 respectively, while immunopurified TDP-43 or A11 myo-granules immunostained with secondary antibodies lack signal (red) (n=3 independent experiments). Scale bar is 1 μm. (e) Complexes immunopurified using TDP-43 or (f) A11 isolated from 5 dpi TA muscle are immunoreactive for A11 and TDP-43 respectively, while immunopurified TDP-43 or A11 myo-granules immunostained with secondary antibodies lack signal (red) (n=3 mice). Scale bar is 0.05 μm. (g) TDP-43 immunopurified complexes isolated from an uninjured TA muscle (contralateral to the 5dpi muscle) reveal no complexes with an A11 oligomeric confirmation (n = 3 mice). Scale bar is 0.05 μm. (h) A11 immunopurified complexes from an uninjured TA muscle (contralateral to the 5dpi muscle) reveal no complexes containing TDP-43 (n = 3 mice). Scale bar is 0.05 μm. (i) Dot blot A11 immunoreactivity in C2C12 cells differentiated into myotubes as compared to myoblasts. Quantification reflects fold change in dot blot signal from myoblast to myotube. Data are mean ± s.d. (n=3 independent experiments) (j) Quantification of dot blot signal for A11 conformation complexes and TDP-43 (k) during skeletal muscle regeneration at 5dpi and at 10dpi compared to contralateral uninjured TA muscle and normalized to HRP-only signal. Quantification reflects fold change in dot blot signal. Data are mean ± s.d., n = 3 mice, p-value are unpaired two-tailed t-test.
    Figure Legend Snippet: Myo-granules containing TDP-43 are amyloid-like oligomers. (Related to Fig 2 ). (a-b) X-ray diffraction on immunoprecipitated myo-granules (right half of both panels) compared to the diffraction of mock IgG immunoprecipitation (left half of panel a) and to the diffraction of super oxide dismutase 1 (SOD1) amyloid oligomers (left half of panel b). In all diffraction patterns two rings at ~4.8 Å and ~10 Å are drawn on the bottom half to highlight absence of an ~4.8 Å reflection in the mock immunoprecipitation and a similar ~4.8 Å reflection with a ~10 Å reflection absence in the SOD1 diffraction. One sample per condition was used. Two diffraction images at different rotations were taken per sample and each image gave similar results. (c) Complexes immunopurified using TDP-43 or (d) A11 isolated from C2C12 myotubes are immunoreactive for A11 and TDP-43 respectively, while immunopurified TDP-43 or A11 myo-granules immunostained with secondary antibodies lack signal (red) (n=3 independent experiments). Scale bar is 1 μm. (e) Complexes immunopurified using TDP-43 or (f) A11 isolated from 5 dpi TA muscle are immunoreactive for A11 and TDP-43 respectively, while immunopurified TDP-43 or A11 myo-granules immunostained with secondary antibodies lack signal (red) (n=3 mice). Scale bar is 0.05 μm. (g) TDP-43 immunopurified complexes isolated from an uninjured TA muscle (contralateral to the 5dpi muscle) reveal no complexes with an A11 oligomeric confirmation (n = 3 mice). Scale bar is 0.05 μm. (h) A11 immunopurified complexes from an uninjured TA muscle (contralateral to the 5dpi muscle) reveal no complexes containing TDP-43 (n = 3 mice). Scale bar is 0.05 μm. (i) Dot blot A11 immunoreactivity in C2C12 cells differentiated into myotubes as compared to myoblasts. Quantification reflects fold change in dot blot signal from myoblast to myotube. Data are mean ± s.d. (n=3 independent experiments) (j) Quantification of dot blot signal for A11 conformation complexes and TDP-43 (k) during skeletal muscle regeneration at 5dpi and at 10dpi compared to contralateral uninjured TA muscle and normalized to HRP-only signal. Quantification reflects fold change in dot blot signal. Data are mean ± s.d., n = 3 mice, p-value are unpaired two-tailed t-test.

    Techniques Used: Immunoprecipitation, Isolation, Mouse Assay, Dot Blot, Two Tailed Test

    TDP-43 binds select sarcomeric mRNA transcripts during muscle formation (Related to Fig 3 ). (a) RNA immunoprecipitation (RIP) from C2C12 myotubes, followed by oligo-dT Northern blot reveals A11 and TDP-43 associate with poly-A RNA (n = 3 biologically independent samples). (b) Schematic of enhanced CLIP (eCLIP) protocol for cultured C2C12 myoblasts and myotubes. (c) Immunoprecipitation of TDP-43 complexes used for eCLIP in C2C12 myoblast (n = 2 biologically independent samples). (d) Same as in (B) but for C2C12 myotubes (n = 2 biologically independent samples). (e) Autoradiogram of 32P-labeled TDP-43 - RNA complexes fractionated by PAGE. White box indicates the area cut and used for eCLIP library preparation (n=1 library prepared per condition). (f) Scatterplots indicate correlation between significant TDP-43 eCLIP peaks in biological replicates. Scatterplot represents fold enrichment for each region in TDP-43 eCLIP relative to paired size matched input (SMInput) with significant peaks in red (p ≤ 10 −8 over SMIput). P-values for each peak to determine significance were calculated by Yates’ Chi-Square test (Perl) or Fisher Exact Test (R computing software) when the expected or observed read number was below five 16 . For myoblasts R values calculated using n = 511137 non-significant peaks and n = 596 significant peaks. For myotubes R values calculated using n = 413368 non-significant peaks and n = 1501 significant peaks. UG-rich motif is significantly enriched in clusters from ORFs and UTRs (p-value determined by DREME software tool). (g) Irreproducible discovery rate (IDR) analysis comparing peak fold enrichment across indicated datasets. (h) TDP-43 eCLIP reveals TDP-43 binds to 3’UTR of TDP-43 transcript in myoblasts (top panel) and myotubes (bottom panel) (n=3 biologically independent experiments giving similar results).
    Figure Legend Snippet: TDP-43 binds select sarcomeric mRNA transcripts during muscle formation (Related to Fig 3 ). (a) RNA immunoprecipitation (RIP) from C2C12 myotubes, followed by oligo-dT Northern blot reveals A11 and TDP-43 associate with poly-A RNA (n = 3 biologically independent samples). (b) Schematic of enhanced CLIP (eCLIP) protocol for cultured C2C12 myoblasts and myotubes. (c) Immunoprecipitation of TDP-43 complexes used for eCLIP in C2C12 myoblast (n = 2 biologically independent samples). (d) Same as in (B) but for C2C12 myotubes (n = 2 biologically independent samples). (e) Autoradiogram of 32P-labeled TDP-43 - RNA complexes fractionated by PAGE. White box indicates the area cut and used for eCLIP library preparation (n=1 library prepared per condition). (f) Scatterplots indicate correlation between significant TDP-43 eCLIP peaks in biological replicates. Scatterplot represents fold enrichment for each region in TDP-43 eCLIP relative to paired size matched input (SMInput) with significant peaks in red (p ≤ 10 −8 over SMIput). P-values for each peak to determine significance were calculated by Yates’ Chi-Square test (Perl) or Fisher Exact Test (R computing software) when the expected or observed read number was below five 16 . For myoblasts R values calculated using n = 511137 non-significant peaks and n = 596 significant peaks. For myotubes R values calculated using n = 413368 non-significant peaks and n = 1501 significant peaks. UG-rich motif is significantly enriched in clusters from ORFs and UTRs (p-value determined by DREME software tool). (g) Irreproducible discovery rate (IDR) analysis comparing peak fold enrichment across indicated datasets. (h) TDP-43 eCLIP reveals TDP-43 binds to 3’UTR of TDP-43 transcript in myoblasts (top panel) and myotubes (bottom panel) (n=3 biologically independent experiments giving similar results).

    Techniques Used: Immunoprecipitation, Northern Blot, Cross-linking Immunoprecipitation, Cell Culture, Labeling, Polyacrylamide Gel Electrophoresis, Software, Significance Assay

    TDP-43 adopts higher-ordered state during normal skeletal muscle formation. (a) Schematic for regeneration of skeletal muscle injuries in wild type mice. (b) TDP-43 immunoreactivity following BaCl 2 -induced tibialis anterior (TA) muscle injury. Embryonic myosin heavy chain (eMHC) in regenerating myofibers with nuclei counterstained with DAPI. Scale bar is 25μm in merged and zoom panels (n = 5 mice per condition providing similar results). (c) Super resolution imaging of TDP-43 immunoreactivity around nascent sarcomeres in the cytoplasm during muscle regeneration. Scale bar is 10μm in merged panels and 5μm in zoom panels. Asterisk identifies an uninjured myofiber lacking eMHC and TDP-43 cytosolic signal. Nuclei are counterstained with DAPI (n=3 biologically independent experiments providing similar results) (d) Quantification of cytoplasmic TDP-43 signal in skeletal muscle myofibers using unpaired two-tailed t-tests for each individual comparison: 5dpi vs UI p-value = 4.36 × 10 −8 (***); 5dpi vs 10dpi p-value = 0.011(*); 10dpi vs UI p-value = 0.015 (not shown) (n=3 biological replicates, n=5 myofibers per replicate). Data are mean ± s.d. (e) Electron microscopy of myo-granules isolated by TDP-43 immunoprecipitation from C2C12 myotubes and from mouse 5dpi TA muscle (n=3 biologically independent experiments providing similar results).
    Figure Legend Snippet: TDP-43 adopts higher-ordered state during normal skeletal muscle formation. (a) Schematic for regeneration of skeletal muscle injuries in wild type mice. (b) TDP-43 immunoreactivity following BaCl 2 -induced tibialis anterior (TA) muscle injury. Embryonic myosin heavy chain (eMHC) in regenerating myofibers with nuclei counterstained with DAPI. Scale bar is 25μm in merged and zoom panels (n = 5 mice per condition providing similar results). (c) Super resolution imaging of TDP-43 immunoreactivity around nascent sarcomeres in the cytoplasm during muscle regeneration. Scale bar is 10μm in merged panels and 5μm in zoom panels. Asterisk identifies an uninjured myofiber lacking eMHC and TDP-43 cytosolic signal. Nuclei are counterstained with DAPI (n=3 biologically independent experiments providing similar results) (d) Quantification of cytoplasmic TDP-43 signal in skeletal muscle myofibers using unpaired two-tailed t-tests for each individual comparison: 5dpi vs UI p-value = 4.36 × 10 −8 (***); 5dpi vs 10dpi p-value = 0.011(*); 10dpi vs UI p-value = 0.015 (not shown) (n=3 biological replicates, n=5 myofibers per replicate). Data are mean ± s.d. (e) Electron microscopy of myo-granules isolated by TDP-43 immunoprecipitation from C2C12 myotubes and from mouse 5dpi TA muscle (n=3 biologically independent experiments providing similar results).

    Techniques Used: Mouse Assay, Imaging, Two Tailed Test, Electron Microscopy, Isolation, Immunoprecipitation

    TDP-43 binds select sarcomeric mRNA transcripts during muscle formation (Related to Fig 3 ). (a) SDS-PAGE gel stained with SYPRO Ruby reveals enrichment for select proteins during fractionation of total cell lysate (T) from C2C12 myotubes, enriched fraction (EF) and TDP-43 immunoprecipitation (IP) (n=3 biologically independent experiments giving similar results). TDP-43 IP and IgG control IP are representative of the fractions used for mass spectrometry. (b) Venn diagram showing significant overlap between the myo-granule proteome and TDP-43 interactome (defined by 22 ) (p-value determined using hypergeometric test). (c) Gene Ontology of myo-granules reveals enrichment for processes relating to the localization and translation of RNA (n=356 proteins, p-value determined using hypergeometric test with Benjamini Hochberg False Discovery Rate (FDR) correction). (d) Venn diagram showing significant overlap between myo-granules and neuronal RNA granule proteomes (defined by 23 ) (p-value determined using hypergeometric test). (e) VCP, a top hit in the myo-granule proteome, colocalizes with cytoplasmic TDP-43 and A11 signal in 5dpi mouse skeletal muscle (n = 3 mice). (f) The RNA-binding protein hnRNPA2B1 is not associated with the myo-granule proteome and remains localized to myonuclei in injured (5 dpi) and uninjured TA muscle (n = 3 mice).
    Figure Legend Snippet: TDP-43 binds select sarcomeric mRNA transcripts during muscle formation (Related to Fig 3 ). (a) SDS-PAGE gel stained with SYPRO Ruby reveals enrichment for select proteins during fractionation of total cell lysate (T) from C2C12 myotubes, enriched fraction (EF) and TDP-43 immunoprecipitation (IP) (n=3 biologically independent experiments giving similar results). TDP-43 IP and IgG control IP are representative of the fractions used for mass spectrometry. (b) Venn diagram showing significant overlap between the myo-granule proteome and TDP-43 interactome (defined by 22 ) (p-value determined using hypergeometric test). (c) Gene Ontology of myo-granules reveals enrichment for processes relating to the localization and translation of RNA (n=356 proteins, p-value determined using hypergeometric test with Benjamini Hochberg False Discovery Rate (FDR) correction). (d) Venn diagram showing significant overlap between myo-granules and neuronal RNA granule proteomes (defined by 23 ) (p-value determined using hypergeometric test). (e) VCP, a top hit in the myo-granule proteome, colocalizes with cytoplasmic TDP-43 and A11 signal in 5dpi mouse skeletal muscle (n = 3 mice). (f) The RNA-binding protein hnRNPA2B1 is not associated with the myo-granule proteome and remains localized to myonuclei in injured (5 dpi) and uninjured TA muscle (n = 3 mice).

    Techniques Used: SDS Page, Staining, Fractionation, Immunoprecipitation, Mass Spectrometry, Significance Assay, Mouse Assay, RNA Binding Assay

    24) Product Images from "GABAA Receptor-Associated Protein Traffics GABAA Receptors to the Plasma Membrane in Neurons"

    Article Title: GABAA Receptor-Associated Protein Traffics GABAA Receptors to the Plasma Membrane in Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3355-04.2004

    Mutations in the γ2-binding domain of YFP-GABARAP disrupt the interaction of YFP-GABARAP with γ2 subunit and NSF in cultured hippocampal neurons. A , Adenovirus was used to direct expression of either wild-type YFP-GABARAP or mutated forms of GABARAP or V5-GABARAP in cultured hippocampal neurons. Western blot of homogenates from cultured neurons using antibody to GABARAP revealed the presence of a 42 kDa band corresponding to wild-type YFP-GABARAP (lane 1), P37A mutated YFP-GABARAP (lane 2), K38A mutated YFP-GABARAP (lane 3), K38A/R40A mutated YFP-GABARAP (lane 4), or V5-GABARAP (lane 6). Cells that did not receive any adenovirus (lane 5) did not have this 42 kDa band. B , Immunoprecipitation of YFP-GABARAP using anti-GFP antibody (or V5-GABARAP using V5 antibody) from neurons infected with wild-type or mutated forms of YFP-GABARAP. Western blot of YFP-GABARAP immunoprecipitate using anti-γ2 antibody shows that YFP-GABARAP (or V5-GABARAP) is capable of pulling down γ2 immunoreactivity. Mutations in the γ2-binding domain reduced the ability of YFP-GABARAP to pull down γ2 immunoreactivity. YFP-GABARAP was also able to pull down NSF immunoreactivity. Mutations in the γ2-binding domain also reduced the ability of YFP-GABARAP to pull down NSF immunoreactivity. C , Quantitation of the amount of immunoprecipitated γ2 or NSF by integration of the optical density of the autoradiograph bands from Western blots revealed that the P37A and K38A/R40A mutants were the most disrupted in there ability to pull down either γ2 or NSF immunoreactivity. Quantities of γ2 or NSF were normalized to the amount of GABARAP detected in each lane.
    Figure Legend Snippet: Mutations in the γ2-binding domain of YFP-GABARAP disrupt the interaction of YFP-GABARAP with γ2 subunit and NSF in cultured hippocampal neurons. A , Adenovirus was used to direct expression of either wild-type YFP-GABARAP or mutated forms of GABARAP or V5-GABARAP in cultured hippocampal neurons. Western blot of homogenates from cultured neurons using antibody to GABARAP revealed the presence of a 42 kDa band corresponding to wild-type YFP-GABARAP (lane 1), P37A mutated YFP-GABARAP (lane 2), K38A mutated YFP-GABARAP (lane 3), K38A/R40A mutated YFP-GABARAP (lane 4), or V5-GABARAP (lane 6). Cells that did not receive any adenovirus (lane 5) did not have this 42 kDa band. B , Immunoprecipitation of YFP-GABARAP using anti-GFP antibody (or V5-GABARAP using V5 antibody) from neurons infected with wild-type or mutated forms of YFP-GABARAP. Western blot of YFP-GABARAP immunoprecipitate using anti-γ2 antibody shows that YFP-GABARAP (or V5-GABARAP) is capable of pulling down γ2 immunoreactivity. Mutations in the γ2-binding domain reduced the ability of YFP-GABARAP to pull down γ2 immunoreactivity. YFP-GABARAP was also able to pull down NSF immunoreactivity. Mutations in the γ2-binding domain also reduced the ability of YFP-GABARAP to pull down NSF immunoreactivity. C , Quantitation of the amount of immunoprecipitated γ2 or NSF by integration of the optical density of the autoradiograph bands from Western blots revealed that the P37A and K38A/R40A mutants were the most disrupted in there ability to pull down either γ2 or NSF immunoreactivity. Quantities of γ2 or NSF were normalized to the amount of GABARAP detected in each lane.

    Techniques Used: Binding Assay, Cell Culture, Expressing, Western Blot, Immunoprecipitation, Infection, Quantitation Assay, Autoradiography

    25) Product Images from "Hydroxyurea-inducible SAR1 gene acts through the Giα/JNK/Jun pathway to regulate γ-globin expression"

    Article Title: Hydroxyurea-inducible SAR1 gene acts through the Giα/JNK/Jun pathway to regulate γ-globin expression

    Journal: Blood

    doi: 10.1182/blood-2013-10-534842

    HU activates NF-κB signaling and enhances NF-κB binding to the SAR1 promoter region. (A) EMSA for the Elk-1/NF-κB was performed using K562 nuclear extracts (10 μg) and oligonucleotide probes containing either a wild-type or mutant Elk-1/NF-κB–binding site. Competition analysis was performed in the presence of 10-, 100-, or 500-fold excess of unlabeled oligonucleotides (right panel). Antibody-supershift assays were performed using antibodies against NF-κB p50, c-Rel, and Elk-1. Two Elk-1/NF-κB–specific DNA-protein complexes are indicated as A and B. The DNA-protein complex supershifted by anti-NF-κB p50 antibody is indicated as ss. (B) EMSA analysis of the effects of HU on NF-κB binding to its recognition site in the SAR1 promoter. EMSA was performed using nuclear extracts (10 μg) isolated from K562 cells treated with 100μM HU for the indicated period and oligonucleotide probes containing the Elk-1/NF-κB–binding site. A and B represent Elk-1/NF-κB–specific DNA-protein complexes as described in A. (C) At day 6 of differentiation, CD34 + cells were treated with or without 100 µM HU. Cells were then harvested and subjected to ChIP assay using antibody against NF-κB or Elk-1 to immunoprecipitate chromatin-protein complexes. A parallel ChIP assay was performed using rabbit IgG for the immunoprecipitation step as a ChIP assay negative control. DNA was amplified and quantitated by PCR with specific primers flanking the SAR1 gene promoter from −137 to −12. (D) CD34 + cells were treated in the presence or absence of 100 μM HU from day 4 to day 7 of differentiation, and preincubated in medium with 0, 5, 7.5, or 10 μg/mL BAY11-7082 for 30 minutes at day 6, transfected with a construct containing the −977 to +49 region of the SAR1 gene, then assayed for luciferase activity 24 hours after transfection. * P
    Figure Legend Snippet: HU activates NF-κB signaling and enhances NF-κB binding to the SAR1 promoter region. (A) EMSA for the Elk-1/NF-κB was performed using K562 nuclear extracts (10 μg) and oligonucleotide probes containing either a wild-type or mutant Elk-1/NF-κB–binding site. Competition analysis was performed in the presence of 10-, 100-, or 500-fold excess of unlabeled oligonucleotides (right panel). Antibody-supershift assays were performed using antibodies against NF-κB p50, c-Rel, and Elk-1. Two Elk-1/NF-κB–specific DNA-protein complexes are indicated as A and B. The DNA-protein complex supershifted by anti-NF-κB p50 antibody is indicated as ss. (B) EMSA analysis of the effects of HU on NF-κB binding to its recognition site in the SAR1 promoter. EMSA was performed using nuclear extracts (10 μg) isolated from K562 cells treated with 100μM HU for the indicated period and oligonucleotide probes containing the Elk-1/NF-κB–binding site. A and B represent Elk-1/NF-κB–specific DNA-protein complexes as described in A. (C) At day 6 of differentiation, CD34 + cells were treated with or without 100 µM HU. Cells were then harvested and subjected to ChIP assay using antibody against NF-κB or Elk-1 to immunoprecipitate chromatin-protein complexes. A parallel ChIP assay was performed using rabbit IgG for the immunoprecipitation step as a ChIP assay negative control. DNA was amplified and quantitated by PCR with specific primers flanking the SAR1 gene promoter from −137 to −12. (D) CD34 + cells were treated in the presence or absence of 100 μM HU from day 4 to day 7 of differentiation, and preincubated in medium with 0, 5, 7.5, or 10 μg/mL BAY11-7082 for 30 minutes at day 6, transfected with a construct containing the −977 to +49 region of the SAR1 gene, then assayed for luciferase activity 24 hours after transfection. * P

    Techniques Used: Binding Assay, Mutagenesis, Isolation, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Amplification, Polymerase Chain Reaction, Transfection, Construct, Luciferase, Activity Assay

    26) Product Images from "Progranulin, a Glycoprotein Deficient in Frontotemporal Dementia, Is a Novel Substrate of Several Protein Disulfide Isomerase Family Proteins"

    Article Title: Progranulin, a Glycoprotein Deficient in Frontotemporal Dementia, Is a Novel Substrate of Several Protein Disulfide Isomerase Family Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026454

    Biochemical identification of PGRN-interacting proteins. ( A ) mPGRN-AP or mPGRN-HA fusion protein constructs were stably transfected into HEK293 cells. Cell lysates or culture medium was analyzed on Western blot with anti-mPGRN antibody. This antibody specifically recognizes mPGRN but not the endogenous hPGRN in HEK293 cells. β-actin was used as the loading control. ( B ) Stably transfected HEK293 cells expressing mPGRN-HA were treated with cross linkers, followed by immunoprecipitation (IP) with HA antibody or control IgG. Immunoisolates were analyzed on Western blot with anti-mPGRN antibody. UT, untransfected HEK293 cells; ST, stably transfected cells.
    Figure Legend Snippet: Biochemical identification of PGRN-interacting proteins. ( A ) mPGRN-AP or mPGRN-HA fusion protein constructs were stably transfected into HEK293 cells. Cell lysates or culture medium was analyzed on Western blot with anti-mPGRN antibody. This antibody specifically recognizes mPGRN but not the endogenous hPGRN in HEK293 cells. β-actin was used as the loading control. ( B ) Stably transfected HEK293 cells expressing mPGRN-HA were treated with cross linkers, followed by immunoprecipitation (IP) with HA antibody or control IgG. Immunoisolates were analyzed on Western blot with anti-mPGRN antibody. UT, untransfected HEK293 cells; ST, stably transfected cells.

    Techniques Used: Construct, Stable Transfection, Transfection, Western Blot, Expressing, Immunoprecipitation

    Biochemical identification of PGRN-interacting proteins. ( A ) Description of the experiment in C . mPGRN-HA stably transfected HEK293 cells were treated with the chemical crosslinker DSS. Immunoprecipitation was performed with an anti-HA antibody. The immunoisolates were analyzed by SDS-PAGE, which was then silver stained. Specific bands were cut out and analyzed by mass spectrometry. ( B ) Image of a gel after silver staining. The identities of bands 1–4 are listed in Table 1 .
    Figure Legend Snippet: Biochemical identification of PGRN-interacting proteins. ( A ) Description of the experiment in C . mPGRN-HA stably transfected HEK293 cells were treated with the chemical crosslinker DSS. Immunoprecipitation was performed with an anti-HA antibody. The immunoisolates were analyzed by SDS-PAGE, which was then silver stained. Specific bands were cut out and analyzed by mass spectrometry. ( B ) Image of a gel after silver staining. The identities of bands 1–4 are listed in Table 1 .

    Techniques Used: Stable Transfection, Transfection, Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Silver Staining

    27) Product Images from "Direct interaction, instrumental for signaling processes, between LacCer and Lyn in the lipid rafts of neutrophil-like cells"

    Article Title: Direct interaction, instrumental for signaling processes, between LacCer and Lyn in the lipid rafts of neutrophil-like cells

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M055319

    Anti-LacCer immunoprecipitation from PNS. PNS from D-HL-60 cells loaded with [ 3 H]LacCer-(N 3 ) were immunoprecipitated with mouse anti-LacCer antibody Huly-m13 or normal mouse IgM. A: The radioactivity associated with the immunoprecipitates was determined
    Figure Legend Snippet: Anti-LacCer immunoprecipitation from PNS. PNS from D-HL-60 cells loaded with [ 3 H]LacCer-(N 3 ) were immunoprecipitated with mouse anti-LacCer antibody Huly-m13 or normal mouse IgM. A: The radioactivity associated with the immunoprecipitates was determined

    Techniques Used: Immunoprecipitation, Radioactivity

    Anti-Lyn immunoprecipitation from D-HL-60 DRM fractions. DRM fractions from cells loaded with [ 3 H]LacCer-(N 3 ) were immunoprecipitated with mouse anti-Lyn IgG or normal mouse IgG. A: The immunoprecipitates were separated by SDS-PAGE and blotted onto PVDF
    Figure Legend Snippet: Anti-Lyn immunoprecipitation from D-HL-60 DRM fractions. DRM fractions from cells loaded with [ 3 H]LacCer-(N 3 ) were immunoprecipitated with mouse anti-Lyn IgG or normal mouse IgG. A: The immunoprecipitates were separated by SDS-PAGE and blotted onto PVDF

    Techniques Used: Immunoprecipitation, SDS Page

    28) Product Images from "Cell cycle stage-specific transcriptional activation of cyclins mediated by HAT2-dependent H4K10 acetylation of promoters in Leishmania donovani"

    Article Title: Cell cycle stage-specific transcriptional activation of cyclins mediated by HAT2-dependent H4K10 acetylation of promoters in Leishmania donovani

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006615

    HAT2 is constitutively nuclear and acetylates H4K10 in vitro . a. Schematic depiction of conserved motifs and residues in the Leishmania donovani HAT2 gene. MOZ/SAS: conserved core catalytic acetyltransferase domain. Cys273 and Glu332: the conserved catalytically important cysteine and glutamate residues. b. Left panel: Western blot analysis of immunoprecipitation reaction from transfectant cells expressing the tagged HAT2-eGFP, performed using anti-eGFP antibodies (1:2000 dilution; 6.75x10 8 cells used per IP reaction). Right panels: IFA of HAT2-eGFP at different cell cycle stages. DAPI: stains DNA compartments; N: nucleus, K: kinetoplast. G1/early S: one nucleus, one short kinetoplast (1N1K); late S/early G2/M: one nucleus, one elongated kinetoplast (1N1K); late G2/M: two nucleii, one kinetoplast (2N1K); post-mitosis—two nucleii, two kinetoplasts (2N2K). Imaging done using a LeicaTCS SP5 confocal microscope, 100X (in oil) objective; analysis using Leica LAS AF software. Magnification bar: 5 μm. c.—e. HAT assays performed with various peptide substrates (sequences in boxes above graphs). Auto: no peptide added. Inset (first panel): western blot analysis of 1/6 pulled-down fraction.
    Figure Legend Snippet: HAT2 is constitutively nuclear and acetylates H4K10 in vitro . a. Schematic depiction of conserved motifs and residues in the Leishmania donovani HAT2 gene. MOZ/SAS: conserved core catalytic acetyltransferase domain. Cys273 and Glu332: the conserved catalytically important cysteine and glutamate residues. b. Left panel: Western blot analysis of immunoprecipitation reaction from transfectant cells expressing the tagged HAT2-eGFP, performed using anti-eGFP antibodies (1:2000 dilution; 6.75x10 8 cells used per IP reaction). Right panels: IFA of HAT2-eGFP at different cell cycle stages. DAPI: stains DNA compartments; N: nucleus, K: kinetoplast. G1/early S: one nucleus, one short kinetoplast (1N1K); late S/early G2/M: one nucleus, one elongated kinetoplast (1N1K); late G2/M: two nucleii, one kinetoplast (2N1K); post-mitosis—two nucleii, two kinetoplasts (2N2K). Imaging done using a LeicaTCS SP5 confocal microscope, 100X (in oil) objective; analysis using Leica LAS AF software. Magnification bar: 5 μm. c.—e. HAT assays performed with various peptide substrates (sequences in boxes above graphs). Auto: no peptide added. Inset (first panel): western blot analysis of 1/6 pulled-down fraction.

    Techniques Used: In Vitro, Western Blot, Immunoprecipitation, Transfection, Expressing, Immunofluorescence, Imaging, Microscopy, Software, HAT Assay

    29) Product Images from "Control of somatic tissue differentiation by the long non-coding RNA TINCR"

    Article Title: Control of somatic tissue differentiation by the long non-coding RNA TINCR

    Journal: Nature

    doi: 10.1038/nature11661

    TINCR interacts with differentiation mRNAs and STAU1 protein a , Enriched GO terms in TINCR-interacting genes detected by RIA-Seq. b , Protein microarray analysis detects TINCR RNA binding to STAU1 protein. Human recombinant protein microarray spotted with approximately 9,400 proteins (left); enlarged 144 protein spot subarray (middle) demonstrating strand-specific binding of TINCR sense strand to STAU1 protein (right); DUPD1 protein negative control is shown. Alexa-Fluor-647-labelled rabbit anti-mouse IgG in the top left corner of each subarray. c , STAU1 protein immunoprecipitation pulls down TINCR RNA. ANCR and LINC1 (also known as XIST ) represent lncRNA controls. d , Streptavidin precipitation of in vitro synthesized biotinylated TINCR RNA pulls down STAU1 protein. HA, haemagglutinin; WB, western blot.
    Figure Legend Snippet: TINCR interacts with differentiation mRNAs and STAU1 protein a , Enriched GO terms in TINCR-interacting genes detected by RIA-Seq. b , Protein microarray analysis detects TINCR RNA binding to STAU1 protein. Human recombinant protein microarray spotted with approximately 9,400 proteins (left); enlarged 144 protein spot subarray (middle) demonstrating strand-specific binding of TINCR sense strand to STAU1 protein (right); DUPD1 protein negative control is shown. Alexa-Fluor-647-labelled rabbit anti-mouse IgG in the top left corner of each subarray. c , STAU1 protein immunoprecipitation pulls down TINCR RNA. ANCR and LINC1 (also known as XIST ) represent lncRNA controls. d , Streptavidin precipitation of in vitro synthesized biotinylated TINCR RNA pulls down STAU1 protein. HA, haemagglutinin; WB, western blot.

    Techniques Used: Microarray, RNA Binding Assay, Recombinant, Binding Assay, Negative Control, Immunoprecipitation, In Vitro, Synthesized, Western Blot

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    Article Snippet: .. Immunoprecipitation and immunoblotting For immunoprecipitation, at 48 h after plasmid transfection, HEK293T cells were washed twice with ice-cold phosphate-buffered saline and then lysed in Pierce immunoprecipitation lysis buffer (Thermo Fisher Scientific; #87787) with protease inhibitor for 20 min at 4 °C. .. Crude lysates were cleared by centrifugation at 20,000×g at 4 °C for 10 min, and the supernatant was incubated with GFP-Trap (ChromoTek, Hauppauge, NY, USA) for 2 h at 4 °C.

    Article Title: Protease-activated receptor 2 signaling modulates susceptibility of colonic epithelium to injury through stabilization of YAP in vivo
    Article Snippet: .. Immunoprecipitation Protein extracts from cultured cells were prepared using immunoprecipitation lysis buffer (Thermo, IL, USA, #87787) with proteinase inhibitor cocktail (Roche) and protein phosphatases inhibitor complex (Applygen). .. The lysates were centrifuged for 10 min at 13,000 rpm at 4 ℃, and supernatants was incubated with anti-YAP1 antibody (Bethyl Laboratories, Inc, TX, USA) or IgG (Santa Cruz, sc-2027) for 2 h, followed by incubation with protein A/G agarose beads (Santa Cruz, sc-2003) overnight.

    Real-time Polymerase Chain Reaction:

    Article Title: An ABCA4 loss-of-function mutation causes a canine form of Stargardt disease
    Article Snippet: .. SDS-Gel Electrophoresis and Western Blotting We extracted protein from the neuroretinal samples of the individuals used in qPCR (see above) by homogenization in Pierce RIPA lysis buffer (Thermo Scientific) supplemented with phosphatase inhibitor cocktail (Sigma, P8340) using the Precellys homogenizer (Bertin Instruments). .. Protein concentration was determined using the Pierce BSA Protein Assay kit (Thermo Fisher Scientific).

    Plasmid Preparation:

    Article Title: Biallelic ERBB3 loss-of-function variants are associated with a novel multisystem syndrome without congenital contracture
    Article Snippet: .. Immunoprecipitation and immunoblotting For immunoprecipitation, at 48 h after plasmid transfection, HEK293T cells were washed twice with ice-cold phosphate-buffered saline and then lysed in Pierce immunoprecipitation lysis buffer (Thermo Fisher Scientific; #87787) with protease inhibitor for 20 min at 4 °C. .. Crude lysates were cleared by centrifugation at 20,000×g at 4 °C for 10 min, and the supernatant was incubated with GFP-Trap (ChromoTek, Hauppauge, NY, USA) for 2 h at 4 °C.

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    Thermo Fisher mild immunoprecipitation buffer
    Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, <t>immunoprecipitation;</t> FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.
    Mild Immunoprecipitation Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The A343D mutation blocks axonal enrichment of surface HA-KCNQ3/KCNQ2 channels. (A) Lysates from HEK293T cells expressing CaM and wild-type KCNQ2 (WT) or mutant KCNQ2 (A343D and R353G) were subjected to <t>immunoprecipitation</t> (IP) with the KCNQ2 antibody. Immunoprecipitation and total cell lysates were analyzed by immunoblotting for KCNQ2 and CaM. β-actin served as a loading control. The A343D mutation abolished whereas the R353G modestly reduced co-immunoprecipitation of CaM with KCNQ2. (B) Representative inverted images of surface HA-KCNQ3 in hippocampal neurons cotransfected with GFP and KCNQ2 WT or mutants (A343D and R353G). The A343D but not the R353G mutation abolished surface expression of HA-KCNQ3/KCNQ2 at the axon, which was identified by immunostaining for the AIS marker, phospho IκBα Ser32 (14D4) ( Figure S4 ). Camera lucida drawings (middle) of neuronal images (upper) show the soma and dendrites (gray) and an axon (black). Pseudo-color images (lower) of the insets in the neuronal images (upper) display differences in the surface HA intensity. Arrows indicate the AIS. Arrowheads mark another axon. Scale bars: 20 µm. (C) The surface “Axon/Dendrite” ratio was reduced by the A343D but not the R353G mutation. The surface AIS/distal axon ratio for A343D mutant channels was not calculated due to their absence at the axonal and AIS surface. (D) Background subtracted, mean intensity of surface HA fluorescence in the AIS, distal axons, soma, and major dendrites. The sample number for each construct was as follows: WT (n = 27), A343D (n = 22), R353G (n = 21), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (*p
    Immunoprecipitation Ip Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, immunoprecipitation; FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.

    Journal: The Journal of Cell Biology

    Article Title: Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

    doi: 10.1083/jcb.201105101

    Figure Lengend Snippet: Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, immunoprecipitation; FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.

    Article Snippet: For lenient binding conditions, a mild immunoprecipitation buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 with protease inhibitor cocktail) was used, and for stringent binding conditions, radioimmunoprecipitation assay buffer (no. 89901; Thermo Fisher Scientific) with protease inhibition cocktail (Roche) was used.

    Techniques: Plasmid Preparation, Construct, Expressing, Generated, Mutagenesis, Western Blot, Activity Assay, In Vivo, Conjugation Assay, Immunoprecipitation, Quantitation Assay, Two Tailed Test

    Hectd1 physically interacts with Hsp90α. (A) Yeast two-hybrid screening of an E11.5 embryonic mouse cDNA library using Hectd1 ANK as bait detected Hsp90α (Gene ID: Hsp90aa1). Two identical clones of Hsp90α consisting of amino acids 241–459 (Hsp90αbd) of the 732–amino acid Hsp90α protein partially overlap with the first charged domain (CD1, amino acids 236–272) and the ATPase domain (amino acids 272–618) of Hsp90α. Black dots indicate locations of Hsp90 peptide fragments found by LC-MS analysis: NPDDITQEEYGEFYK (300–315), TLTIVDTGIGMTK (88–100), KADLINNLGTIAKS (100–113), and GVVDSEDLPLNISR (387–400). Peptides common to Hsp90β include: KEDQTEYLEERR (190–201), RDNSTMGYMMAKK (620–632), and YIDQEELNK (284–292). The C-terminal amino acid sequence MEEVD of Hsp90α is essential for regulated secretion. (B and C) Liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomic screening of Hectd1-binding proteins from E10.5 Hectd1 W embryo head lysates. Hectd1 was immunoprecipitated and associated proteins were resolved by 3–8% Tris-Acetate SDS-PAGE and visualized by Coomassie staining. (B) Individual bands from the Coomassie-stained gel were subjected to tryptic proteolysis, and the resulting peptides were analyzed by LC-MS/MS. (C) Representative MS/MS of a 1,293.54 D peptide. This and six other peptides (dots in A) with high XC scores (3.3–3.7) were identified as belonging to Hsp90α and Hsp90β when searched against the mouse Uniprot protein database using the Sequest algorithm as diagrammed in A. (D) Hsp90αbd binds to Hectd1 ANK in rabbit reticulocyte lysates. In vitro translated, biotinylated Hsp90αbd and Hectd1 ANK were bound and immunoprecipitated using the indicated antibodies and detected by Western blotting with streptavidin-HRP. (E and F) Hectd1 binds to Hsp90 in HEK293T cells. Cells were transfected and immunoprecipitated proteins were subjected to Western blot analyses as indicated. (E) Hsp90αbd binds to Hectd1 ANK in HEK293T cells. (F) Full-length Hsp90 and Hectd1 bind in HEK293T cells. (G) Hsp90 binds to Hectd1 in the developing embryo. Hectd1 was immunoprecipitated from lysates prepared from E12.5 Hectd1 W and Hectd1 O embryo heads and subjected to Western blot analysis as indicated ( n = 4). (H and I) Hsp90 binds to Hectd1 but not the related HECT domain containing Nedd4 Ub ligase. (H and I) HEK293T cells were transfected with HA-Nedd4 or HA-Hectd1 along with Myc-Hsp90. They were then HA immunoprocipitated and subjected to Western blotting (H), or Hsp90 (I) was immunoprecipitated from E12.5 Hectd1 W and Hectd1 O embryo head lysates followed by Western blotting to detect Nedd4 and Hectd1. All data are representative of three independent experiments unless otherwise indicated. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation.

    Journal: The Journal of Cell Biology

    Article Title: Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

    doi: 10.1083/jcb.201105101

    Figure Lengend Snippet: Hectd1 physically interacts with Hsp90α. (A) Yeast two-hybrid screening of an E11.5 embryonic mouse cDNA library using Hectd1 ANK as bait detected Hsp90α (Gene ID: Hsp90aa1). Two identical clones of Hsp90α consisting of amino acids 241–459 (Hsp90αbd) of the 732–amino acid Hsp90α protein partially overlap with the first charged domain (CD1, amino acids 236–272) and the ATPase domain (amino acids 272–618) of Hsp90α. Black dots indicate locations of Hsp90 peptide fragments found by LC-MS analysis: NPDDITQEEYGEFYK (300–315), TLTIVDTGIGMTK (88–100), KADLINNLGTIAKS (100–113), and GVVDSEDLPLNISR (387–400). Peptides common to Hsp90β include: KEDQTEYLEERR (190–201), RDNSTMGYMMAKK (620–632), and YIDQEELNK (284–292). The C-terminal amino acid sequence MEEVD of Hsp90α is essential for regulated secretion. (B and C) Liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomic screening of Hectd1-binding proteins from E10.5 Hectd1 W embryo head lysates. Hectd1 was immunoprecipitated and associated proteins were resolved by 3–8% Tris-Acetate SDS-PAGE and visualized by Coomassie staining. (B) Individual bands from the Coomassie-stained gel were subjected to tryptic proteolysis, and the resulting peptides were analyzed by LC-MS/MS. (C) Representative MS/MS of a 1,293.54 D peptide. This and six other peptides (dots in A) with high XC scores (3.3–3.7) were identified as belonging to Hsp90α and Hsp90β when searched against the mouse Uniprot protein database using the Sequest algorithm as diagrammed in A. (D) Hsp90αbd binds to Hectd1 ANK in rabbit reticulocyte lysates. In vitro translated, biotinylated Hsp90αbd and Hectd1 ANK were bound and immunoprecipitated using the indicated antibodies and detected by Western blotting with streptavidin-HRP. (E and F) Hectd1 binds to Hsp90 in HEK293T cells. Cells were transfected and immunoprecipitated proteins were subjected to Western blot analyses as indicated. (E) Hsp90αbd binds to Hectd1 ANK in HEK293T cells. (F) Full-length Hsp90 and Hectd1 bind in HEK293T cells. (G) Hsp90 binds to Hectd1 in the developing embryo. Hectd1 was immunoprecipitated from lysates prepared from E12.5 Hectd1 W and Hectd1 O embryo heads and subjected to Western blot analysis as indicated ( n = 4). (H and I) Hsp90 binds to Hectd1 but not the related HECT domain containing Nedd4 Ub ligase. (H and I) HEK293T cells were transfected with HA-Nedd4 or HA-Hectd1 along with Myc-Hsp90. They were then HA immunoprocipitated and subjected to Western blotting (H), or Hsp90 (I) was immunoprecipitated from E12.5 Hectd1 W and Hectd1 O embryo head lysates followed by Western blotting to detect Nedd4 and Hectd1. All data are representative of three independent experiments unless otherwise indicated. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation.

    Article Snippet: For lenient binding conditions, a mild immunoprecipitation buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 with protease inhibitor cocktail) was used, and for stringent binding conditions, radioimmunoprecipitation assay buffer (no. 89901; Thermo Fisher Scientific) with protease inhibition cocktail (Roche) was used.

    Techniques: Two Hybrid Screening, cDNA Library Assay, Clone Assay, Liquid Chromatography with Mass Spectroscopy, Sequencing, Liquid Chromatography, Mass Spectrometry, Binding Assay, Immunoprecipitation, SDS Page, Staining, In Vitro, Western Blot, Transfection

    Hectd1 is required for K63-linked Ub n of Hsp90. (A–D) HEK293T cells were transfected, and lysates were subjected to immunoprecipitation and Western blot analysis as indicated. (A) Ubiquitination of Myc-Hsp90 increases with expression of HA-Hectd1 ( n = 2). (B) siRNA-mediated knockdown of endogenous Hectd1 reduces the accumulation of HMW-Hsp90α ( n = 2). (C) Hsp90α ubiquitination utilizes K63 linkages. (D) Hectd1-dependent polyubiquitination of Hsp90 occurs primarily through K63 linkages. (E) HMW Hsp90 species are reduced in Hectd1 mutant heads. E12.5 Hectd1 W (W) and Hectd1 X (X) embryos were cultured in the presence of 10 µM MG132 for 3 h before lysis and immunoprecipitation of Hectd1. Immunoprecipitates were subjected to Western blot analyses to detect Hsp90 that coimmunoprecipitated with Hectd1. (F) Hsp90 ubiquitination is reduced in CM cultures from Hectd1 O (O) and Hectd1 X (X) mutants compared with Hectd1 W (W). Hsp90 was immunoprecipitated from E12.5 CM primary cultures in highly denaturing ubiquitination buffer plus 5% SDS and subjected to Western blot analyses as indicated. The appearance of a 30-kD ubiquitinated protein (asterisk) is reduced in Hectd1 mutant cells. All data are representative of three independent experiments unless otherwise indicated. Abbreviations: W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation; (Ub)n, Ub n ; wt-Ub, wild-type Ub; K48R, mutant Ub lysine 48 arginine; K63R, mutant Ub lysine 48 arginine; K0, lysineless Ub.

    Journal: The Journal of Cell Biology

    Article Title: Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

    doi: 10.1083/jcb.201105101

    Figure Lengend Snippet: Hectd1 is required for K63-linked Ub n of Hsp90. (A–D) HEK293T cells were transfected, and lysates were subjected to immunoprecipitation and Western blot analysis as indicated. (A) Ubiquitination of Myc-Hsp90 increases with expression of HA-Hectd1 ( n = 2). (B) siRNA-mediated knockdown of endogenous Hectd1 reduces the accumulation of HMW-Hsp90α ( n = 2). (C) Hsp90α ubiquitination utilizes K63 linkages. (D) Hectd1-dependent polyubiquitination of Hsp90 occurs primarily through K63 linkages. (E) HMW Hsp90 species are reduced in Hectd1 mutant heads. E12.5 Hectd1 W (W) and Hectd1 X (X) embryos were cultured in the presence of 10 µM MG132 for 3 h before lysis and immunoprecipitation of Hectd1. Immunoprecipitates were subjected to Western blot analyses to detect Hsp90 that coimmunoprecipitated with Hectd1. (F) Hsp90 ubiquitination is reduced in CM cultures from Hectd1 O (O) and Hectd1 X (X) mutants compared with Hectd1 W (W). Hsp90 was immunoprecipitated from E12.5 CM primary cultures in highly denaturing ubiquitination buffer plus 5% SDS and subjected to Western blot analyses as indicated. The appearance of a 30-kD ubiquitinated protein (asterisk) is reduced in Hectd1 mutant cells. All data are representative of three independent experiments unless otherwise indicated. Abbreviations: W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation; (Ub)n, Ub n ; wt-Ub, wild-type Ub; K48R, mutant Ub lysine 48 arginine; K63R, mutant Ub lysine 48 arginine; K0, lysineless Ub.

    Article Snippet: For lenient binding conditions, a mild immunoprecipitation buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 with protease inhibitor cocktail) was used, and for stringent binding conditions, radioimmunoprecipitation assay buffer (no. 89901; Thermo Fisher Scientific) with protease inhibition cocktail (Roche) was used.

    Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Mutagenesis, Cell Culture, Lysis

    The A343D mutation blocks axonal enrichment of surface HA-KCNQ3/KCNQ2 channels. (A) Lysates from HEK293T cells expressing CaM and wild-type KCNQ2 (WT) or mutant KCNQ2 (A343D and R353G) were subjected to immunoprecipitation (IP) with the KCNQ2 antibody. Immunoprecipitation and total cell lysates were analyzed by immunoblotting for KCNQ2 and CaM. β-actin served as a loading control. The A343D mutation abolished whereas the R353G modestly reduced co-immunoprecipitation of CaM with KCNQ2. (B) Representative inverted images of surface HA-KCNQ3 in hippocampal neurons cotransfected with GFP and KCNQ2 WT or mutants (A343D and R353G). The A343D but not the R353G mutation abolished surface expression of HA-KCNQ3/KCNQ2 at the axon, which was identified by immunostaining for the AIS marker, phospho IκBα Ser32 (14D4) ( Figure S4 ). Camera lucida drawings (middle) of neuronal images (upper) show the soma and dendrites (gray) and an axon (black). Pseudo-color images (lower) of the insets in the neuronal images (upper) display differences in the surface HA intensity. Arrows indicate the AIS. Arrowheads mark another axon. Scale bars: 20 µm. (C) The surface “Axon/Dendrite” ratio was reduced by the A343D but not the R353G mutation. The surface AIS/distal axon ratio for A343D mutant channels was not calculated due to their absence at the axonal and AIS surface. (D) Background subtracted, mean intensity of surface HA fluorescence in the AIS, distal axons, soma, and major dendrites. The sample number for each construct was as follows: WT (n = 27), A343D (n = 22), R353G (n = 21), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (*p

    Journal: PLoS ONE

    Article Title: Polarized Axonal Surface Expression of Neuronal KCNQ Potassium Channels Is Regulated by Calmodulin Interaction with KCNQ2 Subunit

    doi: 10.1371/journal.pone.0103655

    Figure Lengend Snippet: The A343D mutation blocks axonal enrichment of surface HA-KCNQ3/KCNQ2 channels. (A) Lysates from HEK293T cells expressing CaM and wild-type KCNQ2 (WT) or mutant KCNQ2 (A343D and R353G) were subjected to immunoprecipitation (IP) with the KCNQ2 antibody. Immunoprecipitation and total cell lysates were analyzed by immunoblotting for KCNQ2 and CaM. β-actin served as a loading control. The A343D mutation abolished whereas the R353G modestly reduced co-immunoprecipitation of CaM with KCNQ2. (B) Representative inverted images of surface HA-KCNQ3 in hippocampal neurons cotransfected with GFP and KCNQ2 WT or mutants (A343D and R353G). The A343D but not the R353G mutation abolished surface expression of HA-KCNQ3/KCNQ2 at the axon, which was identified by immunostaining for the AIS marker, phospho IκBα Ser32 (14D4) ( Figure S4 ). Camera lucida drawings (middle) of neuronal images (upper) show the soma and dendrites (gray) and an axon (black). Pseudo-color images (lower) of the insets in the neuronal images (upper) display differences in the surface HA intensity. Arrows indicate the AIS. Arrowheads mark another axon. Scale bars: 20 µm. (C) The surface “Axon/Dendrite” ratio was reduced by the A343D but not the R353G mutation. The surface AIS/distal axon ratio for A343D mutant channels was not calculated due to their absence at the axonal and AIS surface. (D) Background subtracted, mean intensity of surface HA fluorescence in the AIS, distal axons, soma, and major dendrites. The sample number for each construct was as follows: WT (n = 27), A343D (n = 22), R353G (n = 21), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (*p

    Article Snippet: At 24 hr post transfection, the cells were washed with ice-cold PBS and solubilized in ice-cold immunoprecipitation (IP) buffer containing (in mM): 20 Tris-HCl, 100 NaCl, 2 EDTA, 5 EGTA, 1% Triton X-100 (pH 7.4) supplemented with Halt protease inhibitors (Thermo Fisher Scientific).

    Techniques: Mutagenesis, Expressing, Chick Chorioallantoic Membrane Assay, Immunoprecipitation, Immunostaining, Marker, Fluorescence, Construct

    Mutations in IQ motif abolish axonal enrichment of surface CD4-Q2C. (A) Schematic drawings (not to scale) of a human KCNQ2 subunit (accession #Y15065) and CD4-Q2C showing CaM-binding domain. The amino acids in bold text are critical residues in the CaM-binding consensus IQ motif in helix A. Mutations in the underlined amino acids have been shown to abolish (L339R, I340E, and A343D) or moderately decrease (R353G) CaM interaction with KCNQ2 [24] , [26] . Mutations in the amino acids colored red are associated with BFNC [43] . (B) Lysates from HEK293T cells expressing CaM and CD4-Q2C wild-type (WT) or mutant proteins (L339R, I340E, and A343D) were subjected to immunoprecipitation (IP) with the CD4 antibody. Immunoprecipitation and total cell lysates were analyzed by immunoblotting for CD4 and CaM. β-actin served as a loading control. The L339R, I340E, and A343D mutations abolished co-immunoprecipitation of CaM with CD4-Q2C. (C) Surface immunostaining of CD4-Q2C WT or mutant proteins in hippocampal neurons (DIV 7–8). Camera lucida drawings (lower) of the inverted images of surface CD4-Q2C (upper) show the soma and dendrites (gray) and an axon (black). The axon was identified by immunostaining for the AIS marker phospho IκBα Ser32 (14D4) in the neurons cotransfected with GFP, which allows visualization of all neurites ( Figure S3 ). Arrows indicate the AIS. Scale bars are 20 µm. The L339R, I340E, and A343D mutations abolished surface expression of CD4-Q2C at the AIS and distal axon. (D) The surface “Axon/Dendrite” ratios of CD4-Q2C were reduced to nearly 0 by L339R, I340E, and A343D mutations. (E) Background subtracted, mean intensity of surface CD4 fluorescence in the AIS, distal axons, soma, and major dendrites. AU, arbitrary unit. The sample number for each construct used in (D, E) was as follows: WT (n = 18), L339R (n = 20), I340E (n = 12), A343D (n = 25), and untransfected (n = 20). Ave ± SEM (*p

    Journal: PLoS ONE

    Article Title: Polarized Axonal Surface Expression of Neuronal KCNQ Potassium Channels Is Regulated by Calmodulin Interaction with KCNQ2 Subunit

    doi: 10.1371/journal.pone.0103655

    Figure Lengend Snippet: Mutations in IQ motif abolish axonal enrichment of surface CD4-Q2C. (A) Schematic drawings (not to scale) of a human KCNQ2 subunit (accession #Y15065) and CD4-Q2C showing CaM-binding domain. The amino acids in bold text are critical residues in the CaM-binding consensus IQ motif in helix A. Mutations in the underlined amino acids have been shown to abolish (L339R, I340E, and A343D) or moderately decrease (R353G) CaM interaction with KCNQ2 [24] , [26] . Mutations in the amino acids colored red are associated with BFNC [43] . (B) Lysates from HEK293T cells expressing CaM and CD4-Q2C wild-type (WT) or mutant proteins (L339R, I340E, and A343D) were subjected to immunoprecipitation (IP) with the CD4 antibody. Immunoprecipitation and total cell lysates were analyzed by immunoblotting for CD4 and CaM. β-actin served as a loading control. The L339R, I340E, and A343D mutations abolished co-immunoprecipitation of CaM with CD4-Q2C. (C) Surface immunostaining of CD4-Q2C WT or mutant proteins in hippocampal neurons (DIV 7–8). Camera lucida drawings (lower) of the inverted images of surface CD4-Q2C (upper) show the soma and dendrites (gray) and an axon (black). The axon was identified by immunostaining for the AIS marker phospho IκBα Ser32 (14D4) in the neurons cotransfected with GFP, which allows visualization of all neurites ( Figure S3 ). Arrows indicate the AIS. Scale bars are 20 µm. The L339R, I340E, and A343D mutations abolished surface expression of CD4-Q2C at the AIS and distal axon. (D) The surface “Axon/Dendrite” ratios of CD4-Q2C were reduced to nearly 0 by L339R, I340E, and A343D mutations. (E) Background subtracted, mean intensity of surface CD4 fluorescence in the AIS, distal axons, soma, and major dendrites. AU, arbitrary unit. The sample number for each construct used in (D, E) was as follows: WT (n = 18), L339R (n = 20), I340E (n = 12), A343D (n = 25), and untransfected (n = 20). Ave ± SEM (*p

    Article Snippet: At 24 hr post transfection, the cells were washed with ice-cold PBS and solubilized in ice-cold immunoprecipitation (IP) buffer containing (in mM): 20 Tris-HCl, 100 NaCl, 2 EDTA, 5 EGTA, 1% Triton X-100 (pH 7.4) supplemented with Halt protease inhibitors (Thermo Fisher Scientific).

    Techniques: Chick Chorioallantoic Membrane Assay, Binding Assay, Expressing, Mutagenesis, Immunoprecipitation, Immunostaining, Marker, Fluorescence, Construct

    The BFNC R353G mutation blocks axonal enrichment of surface CD4-Q2C. (A) The R353G mutation reduced but did not abolish co-immunoprecipitation of CaM with CD4-Q2C from transfected HEK293T cells. β-actin served as a loading control for total cell lysates (B) Surface immunostaining of WT or R353G mutant CD4-Q2C in hippocampal neurons (DIV 7–8). Camera lucida drawings (lower) of the inverted images of surface CD4-Q2C (upper) show the soma and dendrites (gray) and an axon (black). The axon was identified by the lack of MAP2 immunostaining in the neurons cotransfected with GFP. The R353G mutation blocked enrichment of CD4-Q2C on the axonal surface by increasing its somatodendritic surface expression. Arrows mark the main axon. Scale bars are 20 µm. (C) In comparison to WT, the surface “Axon/Dendrite” fluorescence ratio of CD4-Q2C was reduced to 1 by the R353G mutation, whereas the surface “AIS/Axon” ratio was unaffected. (D) Background subtracted, mean intensity of surface CD4 fluorescence in the AIS, distal axons, soma, and major dendrites. The R353G mutation increased CD4-Q2C expression at the somatodendritic surface compared to WT. The sample number for each construct used in (C, D) was as follows: WT (n = 23), R353G (n = 18), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (*p

    Journal: PLoS ONE

    Article Title: Polarized Axonal Surface Expression of Neuronal KCNQ Potassium Channels Is Regulated by Calmodulin Interaction with KCNQ2 Subunit

    doi: 10.1371/journal.pone.0103655

    Figure Lengend Snippet: The BFNC R353G mutation blocks axonal enrichment of surface CD4-Q2C. (A) The R353G mutation reduced but did not abolish co-immunoprecipitation of CaM with CD4-Q2C from transfected HEK293T cells. β-actin served as a loading control for total cell lysates (B) Surface immunostaining of WT or R353G mutant CD4-Q2C in hippocampal neurons (DIV 7–8). Camera lucida drawings (lower) of the inverted images of surface CD4-Q2C (upper) show the soma and dendrites (gray) and an axon (black). The axon was identified by the lack of MAP2 immunostaining in the neurons cotransfected with GFP. The R353G mutation blocked enrichment of CD4-Q2C on the axonal surface by increasing its somatodendritic surface expression. Arrows mark the main axon. Scale bars are 20 µm. (C) In comparison to WT, the surface “Axon/Dendrite” fluorescence ratio of CD4-Q2C was reduced to 1 by the R353G mutation, whereas the surface “AIS/Axon” ratio was unaffected. (D) Background subtracted, mean intensity of surface CD4 fluorescence in the AIS, distal axons, soma, and major dendrites. The R353G mutation increased CD4-Q2C expression at the somatodendritic surface compared to WT. The sample number for each construct used in (C, D) was as follows: WT (n = 23), R353G (n = 18), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (*p

    Article Snippet: At 24 hr post transfection, the cells were washed with ice-cold PBS and solubilized in ice-cold immunoprecipitation (IP) buffer containing (in mM): 20 Tris-HCl, 100 NaCl, 2 EDTA, 5 EGTA, 1% Triton X-100 (pH 7.4) supplemented with Halt protease inhibitors (Thermo Fisher Scientific).

    Techniques: Mutagenesis, Immunoprecipitation, Chick Chorioallantoic Membrane Assay, Transfection, Immunostaining, Expressing, Fluorescence, Construct

    Generation and characterization of the NCT-specific single-chain variable fragment. A , immunoprecipitation of NCT full-length (ECD) or truncated (716) ectodomain with NCT-specific Fabs under native condition. Fabs 12, 2, A9, and G9 are NCT-specific Fabs.

    Journal: The Journal of Biological Chemistry

    Article Title: A Synthetic Antibody Fragment Targeting Nicastrin Affects Assembly and Trafficking of γ-Secretase *

    doi: 10.1074/jbc.M114.609636

    Figure Lengend Snippet: Generation and characterization of the NCT-specific single-chain variable fragment. A , immunoprecipitation of NCT full-length (ECD) or truncated (716) ectodomain with NCT-specific Fabs under native condition. Fabs 12, 2, A9, and G9 are NCT-specific Fabs.

    Article Snippet: Cells were then lysed in immunoprecipitation buffer, and biotinylated proteins were captured with NeutrAvidin beads (Thermo Scientific).

    Techniques: Immunoprecipitation

    LOK and SLK are responsible for ezrin/radixin C-terminal phosphorylation in Jeg-3 cells. (A) Enrichment statistics for top four kinases identified by mass spectrometry analysis of ezrin-iFlag cross-linking immunoprecipitates. LOK and SLK were the most highly enriched serine/threonine kinases binding to ezrin-iFlag. (B) LOK-GFP was coexpressed with empty vector or ezrin-iFlag, and cells were subjected to cross-linking immunoprecipitation using anti-Flag (Flag IP). LOK-GFP coimmunoprecipitates with ezrin-iFlag. (C) Cells were treated with the indicated combinations of validated siRNAs against LOK (siLOK1), SLK (siSLK2), or control (siGL2) and Western blotted for LOK to measure knockdown efficiency. The level of phosphorylated ezrin and radixin was determined by Western blotting and quantified by densitometry. (D) Cells were treated with siLOK1 for 3 d and then fixed and stained for ezrin and F-actin. Knockdown of LOK causes loss of microvilli similar to the knockdown of ezrin ( Fig. 3 A ). (E) The presence of microvilli on cells treated with the indicated siRNA was assessed after ezrin and F-actin staining. (F) Cells were treated with DMSO, staurosporine, or erlotinib at the indicated concentrations for 5 min at 37°C, and pERM levels were measured by Western blotting. (G) Cells treated as in F were fixed and stained for ezrin and F-actin. Erlotinib treatment causes a severe reduction in ezrin-containing microvilli. (H) GFP-Flag–tagged kinase constructs were immunoprecipitated using the Flag tag after expression in HEK293T cells and then combined with purified GST-tagged ezrin C terminus in the presence of ATP, which was then subjected to pERM Western blotting. Both LOK and SLK kinase domains are able to phosphorylate the ezrin C-terminal tail in vitro. (I) Cells were cotransfected with an empty vector or GFP fusions of either LOK or SLK along with ezrin-iFlag. The cells were lysed, ezrin-iFlag was immunoprecipitated with anti-Flag, and the phosphorylation of ezrin-iFlag was detected by Western blotting. Both LOK-GFP and SLK-GFP overexpression cause an increase in the overall level of ezrin-iFlag phosphorylation. Data in C, E, and F are means ± SD of three independent experiments. WB, Western blot; Vec, vector. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells

    doi: 10.1083/jcb.201207047

    Figure Lengend Snippet: LOK and SLK are responsible for ezrin/radixin C-terminal phosphorylation in Jeg-3 cells. (A) Enrichment statistics for top four kinases identified by mass spectrometry analysis of ezrin-iFlag cross-linking immunoprecipitates. LOK and SLK were the most highly enriched serine/threonine kinases binding to ezrin-iFlag. (B) LOK-GFP was coexpressed with empty vector or ezrin-iFlag, and cells were subjected to cross-linking immunoprecipitation using anti-Flag (Flag IP). LOK-GFP coimmunoprecipitates with ezrin-iFlag. (C) Cells were treated with the indicated combinations of validated siRNAs against LOK (siLOK1), SLK (siSLK2), or control (siGL2) and Western blotted for LOK to measure knockdown efficiency. The level of phosphorylated ezrin and radixin was determined by Western blotting and quantified by densitometry. (D) Cells were treated with siLOK1 for 3 d and then fixed and stained for ezrin and F-actin. Knockdown of LOK causes loss of microvilli similar to the knockdown of ezrin ( Fig. 3 A ). (E) The presence of microvilli on cells treated with the indicated siRNA was assessed after ezrin and F-actin staining. (F) Cells were treated with DMSO, staurosporine, or erlotinib at the indicated concentrations for 5 min at 37°C, and pERM levels were measured by Western blotting. (G) Cells treated as in F were fixed and stained for ezrin and F-actin. Erlotinib treatment causes a severe reduction in ezrin-containing microvilli. (H) GFP-Flag–tagged kinase constructs were immunoprecipitated using the Flag tag after expression in HEK293T cells and then combined with purified GST-tagged ezrin C terminus in the presence of ATP, which was then subjected to pERM Western blotting. Both LOK and SLK kinase domains are able to phosphorylate the ezrin C-terminal tail in vitro. (I) Cells were cotransfected with an empty vector or GFP fusions of either LOK or SLK along with ezrin-iFlag. The cells were lysed, ezrin-iFlag was immunoprecipitated with anti-Flag, and the phosphorylation of ezrin-iFlag was detected by Western blotting. Both LOK-GFP and SLK-GFP overexpression cause an increase in the overall level of ezrin-iFlag phosphorylation. Data in C, E, and F are means ± SD of three independent experiments. WB, Western blot; Vec, vector. Bars, 10 µm.

    Article Snippet: Lysates were then diluted 10-fold with immunoprecipitation buffer and clarified, and the protein abundance was quantified and adjusted using the 660-nm Protein Assay (Thermo Fisher Scientific), and immunoprecipitated as described in the previous paragraph.

    Techniques: Mass Spectrometry, Binding Assay, Plasmid Preparation, Cross-linking Immunoprecipitation, Western Blot, Staining, Construct, Immunoprecipitation, FLAG-tag, Expressing, Purification, In Vitro, Over Expression