protease inhibitor cocktails  (Thermo Fisher)


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    Structured Review

    Thermo Fisher protease inhibitor cocktails
    Protease Inhibitor Cocktails, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protease inhibitor cocktails/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
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    Negative Control:

    Article Title: CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis
    Article Snippet: RNA immunoprecipitation (RIP) assay HTR-8/SVneo cells were homogenized in RIPA lysis buffer (Elabscience, Wuhan, China) supplemented with protease inhibitor cocktails (Thermo Fisher Scientific). .. Cell lysates were incubated with magnetic bead-coupled anti-Argonaute2 antibody (anti-Ago2, Abcam, Cambridge, UK) or a negative control IgG antibody (Abcam), following the instructions of the Magna RNA Immunoprecipitation Kit (Millipore, Zug, Switzerland).

    Isolation:

    Article Title: CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis
    Article Snippet: RNA immunoprecipitation (RIP) assay HTR-8/SVneo cells were homogenized in RIPA lysis buffer (Elabscience, Wuhan, China) supplemented with protease inhibitor cocktails (Thermo Fisher Scientific). .. Total RNA was isolated from the beads and subjected to qRT-PCR for the enrichment of circZDHHC20, miR-144, and GRHL2.

    Quantitative RT-PCR:

    Article Title: CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis
    Article Snippet: RNA immunoprecipitation (RIP) assay HTR-8/SVneo cells were homogenized in RIPA lysis buffer (Elabscience, Wuhan, China) supplemented with protease inhibitor cocktails (Thermo Fisher Scientific). .. Total RNA was isolated from the beads and subjected to qRT-PCR for the enrichment of circZDHHC20, miR-144, and GRHL2.

    Immunoprecipitation:

    Article Title: CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis
    Article Snippet: .. RNA immunoprecipitation (RIP) assay HTR-8/SVneo cells were homogenized in RIPA lysis buffer (Elabscience, Wuhan, China) supplemented with protease inhibitor cocktails (Thermo Fisher Scientific). .. Cell lysates were incubated with magnetic bead-coupled anti-Argonaute2 antibody (anti-Ago2, Abcam, Cambridge, UK) or a negative control IgG antibody (Abcam), following the instructions of the Magna RNA Immunoprecipitation Kit (Millipore, Zug, Switzerland).

    Incubation:

    Article Title: CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis
    Article Snippet: RNA immunoprecipitation (RIP) assay HTR-8/SVneo cells were homogenized in RIPA lysis buffer (Elabscience, Wuhan, China) supplemented with protease inhibitor cocktails (Thermo Fisher Scientific). .. Cell lysates were incubated with magnetic bead-coupled anti-Argonaute2 antibody (anti-Ago2, Abcam, Cambridge, UK) or a negative control IgG antibody (Abcam), following the instructions of the Magna RNA Immunoprecipitation Kit (Millipore, Zug, Switzerland).

    Protease Inhibitor:

    Article Title: CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis
    Article Snippet: .. RNA immunoprecipitation (RIP) assay HTR-8/SVneo cells were homogenized in RIPA lysis buffer (Elabscience, Wuhan, China) supplemented with protease inhibitor cocktails (Thermo Fisher Scientific). .. Cell lysates were incubated with magnetic bead-coupled anti-Argonaute2 antibody (anti-Ago2, Abcam, Cambridge, UK) or a negative control IgG antibody (Abcam), following the instructions of the Magna RNA Immunoprecipitation Kit (Millipore, Zug, Switzerland).

    Lysis:

    Article Title: CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis
    Article Snippet: .. RNA immunoprecipitation (RIP) assay HTR-8/SVneo cells were homogenized in RIPA lysis buffer (Elabscience, Wuhan, China) supplemented with protease inhibitor cocktails (Thermo Fisher Scientific). .. Cell lysates were incubated with magnetic bead-coupled anti-Argonaute2 antibody (anti-Ago2, Abcam, Cambridge, UK) or a negative control IgG antibody (Abcam), following the instructions of the Magna RNA Immunoprecipitation Kit (Millipore, Zug, Switzerland).

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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: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher immunoprecipitation buffer
    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 <t>immunoprecipitation</t> 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.
    Immunoprecipitation Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher his tagged cour protease inhibitor cocktail
    Identification of the <t>CouR</t> binding site in the <t>couA</t> promoter region by DNase I footprinting. A 439-bp, 6-FAM-labeled DNA fragment (0.45 pmol) was incubated in the presence or absence of CouR (0, 3, and 6 pmol) and p -coumaroyl-CoA (pC-CoA) (2.2 nmol) and
    His Tagged Cour Protease Inhibitor Cocktail, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 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

    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.

    Journal: Disease Models & Mechanisms

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

    doi: 10.1242/dmm.036491

    Figure Lengend 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.

    Article Snippet: Immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl and 0.1% Triton X-100 with protease inhibitor cocktail) was used for less-stringent conditions, while RIPA buffer (Thermo Fisher Scientific, 89901) was used for stringent binding conditions.

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

    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.

    Journal: Disease Models & Mechanisms

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

    doi: 10.1242/dmm.036491

    Figure Lengend 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.

    Article Snippet: Immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl and 0.1% Triton X-100 with protease inhibitor cocktail) was used for less-stringent conditions, while RIPA buffer (Thermo Fisher Scientific, 89901) was used for stringent binding conditions.

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

    Identification of the CouR binding site in the couA promoter region by DNase I footprinting. A 439-bp, 6-FAM-labeled DNA fragment (0.45 pmol) was incubated in the presence or absence of CouR (0, 3, and 6 pmol) and p -coumaroyl-CoA (pC-CoA) (2.2 nmol) and

    Journal: Journal of Bacteriology

    Article Title: Anaerobic p-Coumarate Degradation by Rhodopseudomonas palustris and Identification of CouR, a MarR Repressor Protein That Binds p-Coumaroyl Coenzyme A

    doi: 10.1128/JB.06817-11

    Figure Lengend Snippet: Identification of the CouR binding site in the couA promoter region by DNase I footprinting. A 439-bp, 6-FAM-labeled DNA fragment (0.45 pmol) was incubated in the presence or absence of CouR (0, 3, and 6 pmol) and p -coumaroyl-CoA (pC-CoA) (2.2 nmol) and

    Article Snippet: The cell pellets were suspended in 20 mM Tris (pH 7.9), 500 mM NaCl, 10% glycerol with (for His-tagged CouB) or without (for His-tagged CouA and His-tagged CouR) protease inhibitor cocktail (Thermo Scientific, Santa Clara, CA) and lysed by sonication.

    Techniques: Binding Assay, Footprinting, Labeling, Incubation

    Gel shift assay showing binding of CouR to the couA promoter (A) and the effect of p -coumaroyl-CoA on CouR binding activity (B). (A) CouR protein (0, 2, 4, or 8 pmol) was added to reaction mixtures containing 0.3 pmol of DNA probe. DNA upstream of rpa3929

    Journal: Journal of Bacteriology

    Article Title: Anaerobic p-Coumarate Degradation by Rhodopseudomonas palustris and Identification of CouR, a MarR Repressor Protein That Binds p-Coumaroyl Coenzyme A

    doi: 10.1128/JB.06817-11

    Figure Lengend Snippet: Gel shift assay showing binding of CouR to the couA promoter (A) and the effect of p -coumaroyl-CoA on CouR binding activity (B). (A) CouR protein (0, 2, 4, or 8 pmol) was added to reaction mixtures containing 0.3 pmol of DNA probe. DNA upstream of rpa3929

    Article Snippet: The cell pellets were suspended in 20 mM Tris (pH 7.9), 500 mM NaCl, 10% glycerol with (for His-tagged CouB) or without (for His-tagged CouA and His-tagged CouR) protease inhibitor cocktail (Thermo Scientific, Santa Clara, CA) and lysed by sonication.

    Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay

    The couA promoter sequence and transcriptional start site in the couA upstream region. (A) S1 nuclease analysis of the couA transcript from the wild type and couR mutant. R. palustris cells were grown in PM plus succinate. The couA transcript was detected

    Journal: Journal of Bacteriology

    Article Title: Anaerobic p-Coumarate Degradation by Rhodopseudomonas palustris and Identification of CouR, a MarR Repressor Protein That Binds p-Coumaroyl Coenzyme A

    doi: 10.1128/JB.06817-11

    Figure Lengend Snippet: The couA promoter sequence and transcriptional start site in the couA upstream region. (A) S1 nuclease analysis of the couA transcript from the wild type and couR mutant. R. palustris cells were grown in PM plus succinate. The couA transcript was detected

    Article Snippet: The cell pellets were suspended in 20 mM Tris (pH 7.9), 500 mM NaCl, 10% glycerol with (for His-tagged CouB) or without (for His-tagged CouA and His-tagged CouR) protease inhibitor cocktail (Thermo Scientific, Santa Clara, CA) and lysed by sonication.

    Techniques: Sequencing, Mutagenesis