individual sirna duplexes  (Millipore)


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    Structured Review

    Millipore individual sirna duplexes
    Identification and characterization of the <t>WWP1</t> E3 ligase for PTEN K27-linked polyubiquitination. ( A ) Lysates from DU145 cells transfected with hemagglutinin (HA)–PTEN were immunoprecipitated (IP) with an anti-PTEN antibody followed by mass spectrometric peptide sequencing. WWP1 and PTEN were identified. IgG, immunoglobulin G. ( B ) WWP1 interacts with PTEN endogenously. DU145 cells were immunoprecipitated with either anti-WWP1 or anti-PTEN antibody and then analyzed by Western blot (WB). Input is 5% of the total lysates used in IP. ( C ) Analysis of endogenous PTEN ubiquitination (Ub) in Wwp1 −/− MEFs with stable reconstitution of either WWP1 WT or its catalytically inactive mutant (C890A, abbreviated CA). ( D and E ) Effects of the indicated ubiquitin KR (Lys to Arg) (D) or K-only (E) ubiquitin mutants on WWP1-mediated PTEN polyubiquitination. HEK293T cells were transfected with the indicated constructs, and PTEN ubiquitination was analyzed. The ubiquitinated proteins were pulled down under denaturing conditions by nickel–nitrilotriacetic acid (Ni-NTA) agarose and analyzed by Western blot. ( F ) Analysis of PTEN K27-linked polyubiquitination in PC3 cells expressing the indicated NEDD4 family ubiquitin ligases as in (D). EV, cells transfected with empty vector plasmids. ( G ) In vitro ubiquitination of PTEN by WWP1 E3 ligase. Flag-PTEN purified from HEK293 cells was subject to in vitro ubiquitination reaction in the presence of E1, E2, E3, and ubiquitin or various ubiquitin mutants and then examined by Western blot with anti-PTEN antibody. The input control of WWP1 was determined by Western blotting and is shown at the bottom. ( H ) Analysis of endogenous PTEN ubiquitination by small interfering RNA <t>(siRNA)–mediated</t> WWP1 knock down in U2OS ubiquitin replacement cells treated with 1 μg/ml doxycycline for 48 hours. shUb, shRNAs targeting ubiquitin. ( I ) Effects of the indicated ubiquitin KR mutants on WWP1-mediated PTEN polyubiquitination. PC3 cells were transfected with indicated constructs, and PTEN ubiquitination was analyzed as in (D). In (D), (F), (H), and (I), actin is used as a loading control.
    Individual Sirna Duplexes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway"

    Article Title: Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway

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

    doi: 10.1126/science.aau0159

    Identification and characterization of the WWP1 E3 ligase for PTEN K27-linked polyubiquitination. ( A ) Lysates from DU145 cells transfected with hemagglutinin (HA)–PTEN were immunoprecipitated (IP) with an anti-PTEN antibody followed by mass spectrometric peptide sequencing. WWP1 and PTEN were identified. IgG, immunoglobulin G. ( B ) WWP1 interacts with PTEN endogenously. DU145 cells were immunoprecipitated with either anti-WWP1 or anti-PTEN antibody and then analyzed by Western blot (WB). Input is 5% of the total lysates used in IP. ( C ) Analysis of endogenous PTEN ubiquitination (Ub) in Wwp1 −/− MEFs with stable reconstitution of either WWP1 WT or its catalytically inactive mutant (C890A, abbreviated CA). ( D and E ) Effects of the indicated ubiquitin KR (Lys to Arg) (D) or K-only (E) ubiquitin mutants on WWP1-mediated PTEN polyubiquitination. HEK293T cells were transfected with the indicated constructs, and PTEN ubiquitination was analyzed. The ubiquitinated proteins were pulled down under denaturing conditions by nickel–nitrilotriacetic acid (Ni-NTA) agarose and analyzed by Western blot. ( F ) Analysis of PTEN K27-linked polyubiquitination in PC3 cells expressing the indicated NEDD4 family ubiquitin ligases as in (D). EV, cells transfected with empty vector plasmids. ( G ) In vitro ubiquitination of PTEN by WWP1 E3 ligase. Flag-PTEN purified from HEK293 cells was subject to in vitro ubiquitination reaction in the presence of E1, E2, E3, and ubiquitin or various ubiquitin mutants and then examined by Western blot with anti-PTEN antibody. The input control of WWP1 was determined by Western blotting and is shown at the bottom. ( H ) Analysis of endogenous PTEN ubiquitination by small interfering RNA (siRNA)–mediated WWP1 knock down in U2OS ubiquitin replacement cells treated with 1 μg/ml doxycycline for 48 hours. shUb, shRNAs targeting ubiquitin. ( I ) Effects of the indicated ubiquitin KR mutants on WWP1-mediated PTEN polyubiquitination. PC3 cells were transfected with indicated constructs, and PTEN ubiquitination was analyzed as in (D). In (D), (F), (H), and (I), actin is used as a loading control.
    Figure Legend Snippet: Identification and characterization of the WWP1 E3 ligase for PTEN K27-linked polyubiquitination. ( A ) Lysates from DU145 cells transfected with hemagglutinin (HA)–PTEN were immunoprecipitated (IP) with an anti-PTEN antibody followed by mass spectrometric peptide sequencing. WWP1 and PTEN were identified. IgG, immunoglobulin G. ( B ) WWP1 interacts with PTEN endogenously. DU145 cells were immunoprecipitated with either anti-WWP1 or anti-PTEN antibody and then analyzed by Western blot (WB). Input is 5% of the total lysates used in IP. ( C ) Analysis of endogenous PTEN ubiquitination (Ub) in Wwp1 −/− MEFs with stable reconstitution of either WWP1 WT or its catalytically inactive mutant (C890A, abbreviated CA). ( D and E ) Effects of the indicated ubiquitin KR (Lys to Arg) (D) or K-only (E) ubiquitin mutants on WWP1-mediated PTEN polyubiquitination. HEK293T cells were transfected with the indicated constructs, and PTEN ubiquitination was analyzed. The ubiquitinated proteins were pulled down under denaturing conditions by nickel–nitrilotriacetic acid (Ni-NTA) agarose and analyzed by Western blot. ( F ) Analysis of PTEN K27-linked polyubiquitination in PC3 cells expressing the indicated NEDD4 family ubiquitin ligases as in (D). EV, cells transfected with empty vector plasmids. ( G ) In vitro ubiquitination of PTEN by WWP1 E3 ligase. Flag-PTEN purified from HEK293 cells was subject to in vitro ubiquitination reaction in the presence of E1, E2, E3, and ubiquitin or various ubiquitin mutants and then examined by Western blot with anti-PTEN antibody. The input control of WWP1 was determined by Western blotting and is shown at the bottom. ( H ) Analysis of endogenous PTEN ubiquitination by small interfering RNA (siRNA)–mediated WWP1 knock down in U2OS ubiquitin replacement cells treated with 1 μg/ml doxycycline for 48 hours. shUb, shRNAs targeting ubiquitin. ( I ) Effects of the indicated ubiquitin KR mutants on WWP1-mediated PTEN polyubiquitination. PC3 cells were transfected with indicated constructs, and PTEN ubiquitination was analyzed as in (D). In (D), (F), (H), and (I), actin is used as a loading control.

    Techniques Used: Transfection, Immunoprecipitation, Sequencing, Western Blot, Mutagenesis, Construct, Expressing, Plasmid Preparation, In Vitro, Purification, Small Interfering RNA

    Related Articles

    Western Blot:

    Article Title: Cross-talk among writers, readers, and erasers of m6A regulates cancer growth and progression
    Article Snippet: .. Cancer cell lines were transfected with scrambled-siRNA or METTL14-siRNA/ALKBH5-siRNA (Sigma-Aldrich) for 48 or 72 hours before they were subjected to qRT-PCR or Western blot analysis, as described previously ( ). .. Antibodies against ALKBH5 (#HPA 007196), β-actin (#A3854), GAPDH (#G9295), and METTL14 (#HPA038002) were purchased from Sigma-Aldrich.

    Transfection:

    Article Title: MICU1 drives glycolysis and chemoresistance in ovarian cancer
    Article Snippet: .. Plasmids and siRNA transfection Gene silencing was performed for CP20 and OV90 cell lines in 6 cm culture dish containing 5 × 105 cells using Hiperfect (Qiagen) and commercially validated 10 μM siRNA (scrambled control siRNA (#1027280), QIAGEN, CA, USA) or siRNA against human MICU1 (SASI_Hs01_00070243, SASI_Hs01_00070249 Sigma) (# S1041403600 Qiagen) in OPTIMEM (Invitrogen). .. Effective silencing was achieved after 48-72 h of transfection (determined by protein expression) and all experiments with gene silencing were performed 48-96 h post transfection.

    Article Title: Cross-talk among writers, readers, and erasers of m6A regulates cancer growth and progression
    Article Snippet: .. Cancer cell lines were transfected with scrambled-siRNA or METTL14-siRNA/ALKBH5-siRNA (Sigma-Aldrich) for 48 or 72 hours before they were subjected to qRT-PCR or Western blot analysis, as described previously ( ). .. Antibodies against ALKBH5 (#HPA 007196), β-actin (#A3854), GAPDH (#G9295), and METTL14 (#HPA038002) were purchased from Sigma-Aldrich.

    Negative Control:

    Article Title: Combining peptide TNIIIA2 with all-trans retinoic acid accelerates N-Myc protein degradation and neuronal differentiation in MYCN-amplified neuroblastoma cells
    Article Snippet: .. 200 μL of Opti-MEM (Invitrogen) including talin small interference RNA (siRNA) (Sigma) or siPerfect Negative Control (Sigma) and 2 μL of Lipofectamine RNAi Max (Invitrogen) were mixed and placed still for 20 minutes at room temperature. .. 1 mL of IMR-32 cell suspension (6 × 104 cells/mL) was added to the solution and the mixture was incubated in a 5% CO2 incubator at 37°C for 48 hours.

    Quantitative RT-PCR:

    Article Title: Cross-talk among writers, readers, and erasers of m6A regulates cancer growth and progression
    Article Snippet: .. Cancer cell lines were transfected with scrambled-siRNA or METTL14-siRNA/ALKBH5-siRNA (Sigma-Aldrich) for 48 or 72 hours before they were subjected to qRT-PCR or Western blot analysis, as described previously ( ). .. Antibodies against ALKBH5 (#HPA 007196), β-actin (#A3854), GAPDH (#G9295), and METTL14 (#HPA038002) were purchased from Sigma-Aldrich.

    Lysis:

    Article Title: Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc–siRNA conjugates
    Article Snippet: .. Ago2-bound siRNA from mouse liver was quantified by preparing liver lysates at 100 mg/ml in lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2mM EDTA, 0.5% Triton-X 100) supplemented with freshly added protease inhibitors (Sigma-Aldrich, P8340) at 1:100 dilution and 1 mM PMSF (Life Technologies). .. Total liver lysate (10 mg) was used for each Ago2 immunoprecipitation (IP) and control IP.

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    Millipore immunoblot analysis immunoblotting
    BMI1 suppresses cell death by stabilizing cyclin E1. a , <t>Immunoblot</t> analysis of cyclins and CDKs in pooled BMI1-sensitive clones following BMI1 knockdown. α-tubulin levels are shown as loading control. b , Immunoblot analysis of cyclin E1 levels in BE(2)-C cells either uninfected (parental) or infected with retroviruses expressing shRNA sequences against GFP (GFPsh) or different regions of CCNE1 (CCNE1sh-80 and -81). Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the cyclin E1 level in parental cells. c , Crystal violet staining of BE(2)-C cells expressing either GFPsh or CCNE1sh-80. d , Immunoblot analysis of BMI1 and cyclin E1 levels in pooled BMI1-sensitive clones infected with retroviruses expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 3 days. α-tubulin levels are shown as loading control. e , Phase contrast imaging of pooled BMI1-sensitive clones expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 6 days. Scale bars, 100 μm. f , qRT-PCR analysis of CCND1 and CCNE1 mRNA levels in pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days (error bars, s.d., n=3). g , Quantification of cyclin E1 half-life in pooled BMI1-sensitive clones cultured in the presence or absence of Doxy for 3 days. Samples were collected at various time points following addition of cycloheximide (CHX) for immunoblot analysis. Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the initial levels at time zero (error bars, s.d., n=4). h , In vivo ubiquitination assay of pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days and co-transfected with Flag-ubiquitin and Myc-cyclin E1 expression plasmids. Polyubiquitinated cyclin E1 was detected by immunoprecipitation of Myccyclin E1, followed by <t>immunoblotting</t> for Flag-ubiquitin.
    Immunoblot Analysis Immunoblotting, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BMI1 suppresses cell death by stabilizing cyclin E1. a , Immunoblot analysis of cyclins and CDKs in pooled BMI1-sensitive clones following BMI1 knockdown. α-tubulin levels are shown as loading control. b , Immunoblot analysis of cyclin E1 levels in BE(2)-C cells either uninfected (parental) or infected with retroviruses expressing shRNA sequences against GFP (GFPsh) or different regions of CCNE1 (CCNE1sh-80 and -81). Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the cyclin E1 level in parental cells. c , Crystal violet staining of BE(2)-C cells expressing either GFPsh or CCNE1sh-80. d , Immunoblot analysis of BMI1 and cyclin E1 levels in pooled BMI1-sensitive clones infected with retroviruses expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 3 days. α-tubulin levels are shown as loading control. e , Phase contrast imaging of pooled BMI1-sensitive clones expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 6 days. Scale bars, 100 μm. f , qRT-PCR analysis of CCND1 and CCNE1 mRNA levels in pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days (error bars, s.d., n=3). g , Quantification of cyclin E1 half-life in pooled BMI1-sensitive clones cultured in the presence or absence of Doxy for 3 days. Samples were collected at various time points following addition of cycloheximide (CHX) for immunoblot analysis. Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the initial levels at time zero (error bars, s.d., n=4). h , In vivo ubiquitination assay of pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days and co-transfected with Flag-ubiquitin and Myc-cyclin E1 expression plasmids. Polyubiquitinated cyclin E1 was detected by immunoprecipitation of Myccyclin E1, followed by immunoblotting for Flag-ubiquitin.

    Journal: Oncogene

    Article Title: Cyclin E1 is a common target of BMI1 and MYCN and a prognostic marker for neuroblastoma progression

    doi: 10.1038/onc.2011.536

    Figure Lengend Snippet: BMI1 suppresses cell death by stabilizing cyclin E1. a , Immunoblot analysis of cyclins and CDKs in pooled BMI1-sensitive clones following BMI1 knockdown. α-tubulin levels are shown as loading control. b , Immunoblot analysis of cyclin E1 levels in BE(2)-C cells either uninfected (parental) or infected with retroviruses expressing shRNA sequences against GFP (GFPsh) or different regions of CCNE1 (CCNE1sh-80 and -81). Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the cyclin E1 level in parental cells. c , Crystal violet staining of BE(2)-C cells expressing either GFPsh or CCNE1sh-80. d , Immunoblot analysis of BMI1 and cyclin E1 levels in pooled BMI1-sensitive clones infected with retroviruses expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 3 days. α-tubulin levels are shown as loading control. e , Phase contrast imaging of pooled BMI1-sensitive clones expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 6 days. Scale bars, 100 μm. f , qRT-PCR analysis of CCND1 and CCNE1 mRNA levels in pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days (error bars, s.d., n=3). g , Quantification of cyclin E1 half-life in pooled BMI1-sensitive clones cultured in the presence or absence of Doxy for 3 days. Samples were collected at various time points following addition of cycloheximide (CHX) for immunoblot analysis. Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the initial levels at time zero (error bars, s.d., n=4). h , In vivo ubiquitination assay of pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days and co-transfected with Flag-ubiquitin and Myc-cyclin E1 expression plasmids. Polyubiquitinated cyclin E1 was detected by immunoprecipitation of Myccyclin E1, followed by immunoblotting for Flag-ubiquitin.

    Article Snippet: Immunoblot analysis Immunoblotting was conducted according to standard procedures using the following primary antibodies: mouse anti-BMI1 (05-637, 1:500; Upstate, Millipore, Billerica, MA), rabbit anti-cyclin A2 (sc-751, 1:200), mouse anti-cyclin B1 (sc-245, 1:200), mouse anti-cyclin D1 (sc-20044, 1:200), mouse anti-cyclin E1 (sc-56310, 1:200), rabbit anti-cyclin E2 (ab40890, 1:2000; Abcam, Cambridge, MA), mouse anti-Flag tag (F-4042, 1:2000; Sigma-Aldrich), mouse anti-MYCN (OP13, 1:200; Calbiochem, EMD Biosciences, San Diego, CA), mouse anti-Myc tag (9E10, hybridoma supernatant, 1:10), rabbit anti-p14ARF (A300-340A, 1:500; Bethyl Laboratories, Montgomery, TX), rabbit anti-p16Ink4a (sc-468, 1:200), rabbit anti-β-actin (600-401-886, 1:2000; Rockland Immunochemicals, Gilbertsville, PA), and mouse anti-α-tubulin (B-5-1-2, 1:5000; Sigma-Aldrich).

    Techniques: Clone Assay, Infection, Expressing, shRNA, Staining, Cell Culture, Imaging, Quantitative RT-PCR, In Vivo, Ubiquitin Assay, Transfection, Immunoprecipitation