individual sirna duplexes Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    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 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual sirna duplexes/product/Millipore
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    individual sirna duplexes - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    88
    Horizon Discovery individual sirna duplexes
    Confirmation that SF3A1 and GOLGA4 regulate LPS-induced IL-6 production using a second mouse macrophage cell line. A and B, indicated pools of <t>siRNA</t> duplexes were <t>transfected</t> into the RAW264.7 mouse macrophage cell line; cells were simulated with LPS
    Individual Sirna Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 88/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual sirna duplexes/product/Horizon Discovery
    Average 88 stars, based on 63 article reviews
    Price from $9.99 to $1999.99
    individual sirna duplexes - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    88
    Thermo Fisher individual sirna duplexes
    The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or <t>Cdc42</t> <t>siRNA</t> was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.
    Individual Sirna Duplexes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual sirna duplexes/product/Thermo Fisher
    Average 88 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    individual sirna duplexes - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    85
    Thermo Fisher sirnas individual duplex sirnas
    The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or <t>Cdc42</t> <t>siRNA</t> was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.
    Sirnas Individual Duplex Sirnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirnas individual duplex sirnas/product/Thermo Fisher
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sirnas individual duplex sirnas - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    85
    Horizon Discovery rna interference individual sirna duplexes
    Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin <t>RNA</t> <t>(shRNA)</t> sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P
    Rna Interference Individual Sirna Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna interference individual sirna duplexes/product/Horizon Discovery
    Average 85 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    rna interference individual sirna duplexes - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    85
    Horizon Discovery individual gene specific sirna duplexes
    Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin <t>RNA</t> <t>(shRNA)</t> sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P
    Individual Gene Specific Sirna Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual gene specific sirna duplexes/product/Horizon Discovery
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    individual gene specific sirna duplexes - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    85
    Horizon Discovery individual sirnas duplex dharmacon
    Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin <t>RNA</t> <t>(shRNA)</t> sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P
    Individual Sirnas Duplex Dharmacon, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual sirnas duplex dharmacon/product/Horizon Discovery
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    individual sirnas duplex dharmacon - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    85
    Horizon Discovery sigenome individual duplex sirnas
    Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin <t>RNA</t> <t>(shRNA)</t> sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P
    Sigenome Individual Duplex Sirnas, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sigenome individual duplex sirnas/product/Horizon Discovery
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sigenome individual duplex sirnas - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    Image Search Results


    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.

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

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

    doi: 10.1126/science.aau0159

    Figure Lengend 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.

    Article Snippet: The two individual siRNA duplexes targeted to WWP1 and control nontarget siRNA were purchased from Sigma Aldrich, whereas siRNA SMARTpool targeted to MYC was purchased from Dharmacon.

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

    mRNA expression of NMIIA, NMIIB, and NMIIC in two different samples of WJ-MSCs, as analyzed by semiquantitative RT-PCR using isoform-specific primers. 18s rRNA was used as an internal control (A) . Protein expression of NMIIC was analyzed in WJ-MSCs. The MCF-7 cell line was used as positive control. The same blot was reprobed with the anti-GAPDH antibody to demonstrate equal loading (B) . Localization of NMIIA and NMIIB in cultured WJ-MSCs. Representative confocal images of cultured WJ-MSCs immunostained for NMII A and NMIIB ( C , F , respectively ) , phalloidin-stained F-actin ( D , G) , merged (E , H) and mesenchymal marker vimentin ( I) are presented. Nuclei are stained with DAPI. A negative control with the omission of incubation with primary antibody is shown (J) . Scale bar: 50 μm. Results are representative of at least two independent biological or donor samples. MSCs, mesenchymal stem cells; NMII, nonmuscle myosin II; RT-PCR, reverse transcription-polymerase chain reaction; WJ, Wharton's jelly; WJ-MSCs, WJ-derived MSCs.

    Journal: Stem Cells and Development

    Article Title: Role of Nonmuscle Myosin II in Migration of Wharton's Jelly-Derived Mesenchymal Stem Cells

    doi: 10.1089/scd.2015.0095

    Figure Lengend Snippet: mRNA expression of NMIIA, NMIIB, and NMIIC in two different samples of WJ-MSCs, as analyzed by semiquantitative RT-PCR using isoform-specific primers. 18s rRNA was used as an internal control (A) . Protein expression of NMIIC was analyzed in WJ-MSCs. The MCF-7 cell line was used as positive control. The same blot was reprobed with the anti-GAPDH antibody to demonstrate equal loading (B) . Localization of NMIIA and NMIIB in cultured WJ-MSCs. Representative confocal images of cultured WJ-MSCs immunostained for NMII A and NMIIB ( C , F , respectively ) , phalloidin-stained F-actin ( D , G) , merged (E , H) and mesenchymal marker vimentin ( I) are presented. Nuclei are stained with DAPI. A negative control with the omission of incubation with primary antibody is shown (J) . Scale bar: 50 μm. Results are representative of at least two independent biological or donor samples. MSCs, mesenchymal stem cells; NMII, nonmuscle myosin II; RT-PCR, reverse transcription-polymerase chain reaction; WJ, Wharton's jelly; WJ-MSCs, WJ-derived MSCs.

    Article Snippet: Isoform-specific individual siRNA duplexes against NMIIA and NMIIB (Sigma) were used at a final concentration of 20 nM, and the MISSION siRNA Universal Negative Control (Sigma) was used at the same concentration in the control experiments.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Cell Culture, Staining, Marker, Negative Control, Incubation, Derivative Assay

    SiRNA-mediated downregulation of NMII isoforms. Western blots of WJ-MSC lysates, prepared 72 h post-transfection, showed selective downregulation of NMIIA and NMIIB protein expression by siRNA specific for NMIIA or NMIIB isoforms, respectively. The same blot was reprobed with anti-GAPDH antibody to demonstrate equal loading (A) . Morphology of WJ-MSCs 72 h after transfection with negative control siRNA, NMIIA siRNA, NMIIB SiRNA, or cotransfection with NMIIA plus NMIIB siRNA. SiRNA-mediated downregulation of NMIIA, but not NMIIB, resulted in extended tails as shown by white arrows . Boxed regions are zoomed. Scale bar: 200 μm (B) . Scratch migration assay of siRNA-transfected WJ-MSCs. WJ-MSCs were seeded in 12-well plates and transfected with negative control siRNA or siRNAs against NMIIA, NMIIB, and both NMIIA and NMIIB using Lipofectamine 3000. Seventy-two hours after siRNA transfection, confluent monolayer cultures were wounded with a sterile pipette tip. Images of the wound healing were captured at 0 h, 5, and 12 h (C) . Scale bar: 200 μm. Results are representative of at least three independent biological or donor samples.

    Journal: Stem Cells and Development

    Article Title: Role of Nonmuscle Myosin II in Migration of Wharton's Jelly-Derived Mesenchymal Stem Cells

    doi: 10.1089/scd.2015.0095

    Figure Lengend Snippet: SiRNA-mediated downregulation of NMII isoforms. Western blots of WJ-MSC lysates, prepared 72 h post-transfection, showed selective downregulation of NMIIA and NMIIB protein expression by siRNA specific for NMIIA or NMIIB isoforms, respectively. The same blot was reprobed with anti-GAPDH antibody to demonstrate equal loading (A) . Morphology of WJ-MSCs 72 h after transfection with negative control siRNA, NMIIA siRNA, NMIIB SiRNA, or cotransfection with NMIIA plus NMIIB siRNA. SiRNA-mediated downregulation of NMIIA, but not NMIIB, resulted in extended tails as shown by white arrows . Boxed regions are zoomed. Scale bar: 200 μm (B) . Scratch migration assay of siRNA-transfected WJ-MSCs. WJ-MSCs were seeded in 12-well plates and transfected with negative control siRNA or siRNAs against NMIIA, NMIIB, and both NMIIA and NMIIB using Lipofectamine 3000. Seventy-two hours after siRNA transfection, confluent monolayer cultures were wounded with a sterile pipette tip. Images of the wound healing were captured at 0 h, 5, and 12 h (C) . Scale bar: 200 μm. Results are representative of at least three independent biological or donor samples.

    Article Snippet: Isoform-specific individual siRNA duplexes against NMIIA and NMIIB (Sigma) were used at a final concentration of 20 nM, and the MISSION siRNA Universal Negative Control (Sigma) was used at the same concentration in the control experiments.

    Techniques: Western Blot, Transfection, Expressing, Negative Control, Cotransfection, Migration, Transferring

    Confirmation that SF3A1 and GOLGA4 regulate LPS-induced IL-6 production using a second mouse macrophage cell line. A and B, indicated pools of siRNA duplexes were transfected into the RAW264.7 mouse macrophage cell line; cells were simulated with LPS

    Journal: The Journal of Biological Chemistry

    Article Title: An Evolutionarily Conserved Innate Immunity Protein Interaction Network *

    doi: 10.1074/jbc.M112.407205

    Figure Lengend Snippet: Confirmation that SF3A1 and GOLGA4 regulate LPS-induced IL-6 production using a second mouse macrophage cell line. A and B, indicated pools of siRNA duplexes were transfected into the RAW264.7 mouse macrophage cell line; cells were simulated with LPS

    Article Snippet: In brief, pools of four siRNA duplexes or individual siRNA duplexes (Dharmacon) were transfected into either of two mouse macrophage cell lines (J774A.1 or RAW264.7) using the Amaxa nucleofector 96-well shuttle according to the manufacturer's instructions.

    Techniques: Transfection

    Most genes in the innate immunity protein interaction network affect LPS-induced IL-6 production in the J774A.1 mouse macrophage cell line. A, pools of four siRNA duplexes per gene were transfected into the mouse macrophage cell line J774A.1; cells were

    Journal: The Journal of Biological Chemistry

    Article Title: An Evolutionarily Conserved Innate Immunity Protein Interaction Network *

    doi: 10.1074/jbc.M112.407205

    Figure Lengend Snippet: Most genes in the innate immunity protein interaction network affect LPS-induced IL-6 production in the J774A.1 mouse macrophage cell line. A, pools of four siRNA duplexes per gene were transfected into the mouse macrophage cell line J774A.1; cells were

    Article Snippet: In brief, pools of four siRNA duplexes or individual siRNA duplexes (Dharmacon) were transfected into either of two mouse macrophage cell lines (J774A.1 or RAW264.7) using the Amaxa nucleofector 96-well shuttle according to the manufacturer's instructions.

    Techniques: Transfection

    MACF1 inhibits PAMP-induced cytokine production in RAW264.7 cells and in vivo . A, pool of four siRNA duplexes targeting either Macf1 or a nontargeting control siRNA duplex pool were transfected into the mouse macrophage cell line RAW264.7; cells were

    Journal: The Journal of Biological Chemistry

    Article Title: An Evolutionarily Conserved Innate Immunity Protein Interaction Network *

    doi: 10.1074/jbc.M112.407205

    Figure Lengend Snippet: MACF1 inhibits PAMP-induced cytokine production in RAW264.7 cells and in vivo . A, pool of four siRNA duplexes targeting either Macf1 or a nontargeting control siRNA duplex pool were transfected into the mouse macrophage cell line RAW264.7; cells were

    Article Snippet: In brief, pools of four siRNA duplexes or individual siRNA duplexes (Dharmacon) were transfected into either of two mouse macrophage cell lines (J774A.1 or RAW264.7) using the Amaxa nucleofector 96-well shuttle according to the manufacturer's instructions.

    Techniques: In Vivo, Transfection

    The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.

    Journal: The Journal of Cell Biology

    Article Title: Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis

    doi: 10.1083/jcb.200807121

    Figure Lengend Snippet: The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.

    Article Snippet: RNAi To deplete Cdc42, a SMARTpool (a mixture of four siRNA duplexes) and individual siRNA duplexes were purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Staining, Quantitation Assay

    Cdc42 depletion disrupts mitotic spindle orientation. (A) Diagram depicting spindle angle measurement. The centroid of the cyst (dark blue circle) and the center of the spindle axis (pink circles) of a metaphase cell were drawn using ImageJ. The angle (red) between the spindle axis (black lines) and the line connecting the centroid of the cyst to the center of the spindle (dashed lines) was determined. To analyze spindle poles in different z sections, three z sections were taken so as to include both spindle poles and were merged as shown. Three schematic spindles are shown. The right and middle spindle examples represent correctly oriented spindles whose poles are positioned in one section (z2; middle spindle) or in different sections (z1 and z3; right spindle). The left spindle represents a misoriented spindle whose poles are in different z sections. Spindle microtubules, green; centrosomes, yellow; DNA, light blue. (B) Scatter diagram of metaphase spindle angles in cysts that were transfected with control or two Cdc42 siRNA duplexes from three independent experiments. Pink circles indicate mean values, green circles indicate individual data points, and error bars represent the SEM of the total number of spindles analyzed (N). (C) Caco-2 was transfected with control or Cdc42 siRNA and was fixed and stained for DNA (blue), tubulin (green), and filamentous actin (F-actin; red). Single confocal sections through the center of the cysts are shown. Three z sections are shown for the Cdc42 siRNA cyst to reveal both poles of the misoriented spindle. (D and E) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue) and aPKC (green). (D) Cdc42 siRNA structures contain cells in the middle, with apical domains present between inner and outer cells. (E) Cdc42 siRNA structures possess apical domains that are not in the center and single cells with more than one apical domain. Arrows indicate cells with multiple distinct apical patches on their surface. Bars, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis

    doi: 10.1083/jcb.200807121

    Figure Lengend Snippet: Cdc42 depletion disrupts mitotic spindle orientation. (A) Diagram depicting spindle angle measurement. The centroid of the cyst (dark blue circle) and the center of the spindle axis (pink circles) of a metaphase cell were drawn using ImageJ. The angle (red) between the spindle axis (black lines) and the line connecting the centroid of the cyst to the center of the spindle (dashed lines) was determined. To analyze spindle poles in different z sections, three z sections were taken so as to include both spindle poles and were merged as shown. Three schematic spindles are shown. The right and middle spindle examples represent correctly oriented spindles whose poles are positioned in one section (z2; middle spindle) or in different sections (z1 and z3; right spindle). The left spindle represents a misoriented spindle whose poles are in different z sections. Spindle microtubules, green; centrosomes, yellow; DNA, light blue. (B) Scatter diagram of metaphase spindle angles in cysts that were transfected with control or two Cdc42 siRNA duplexes from three independent experiments. Pink circles indicate mean values, green circles indicate individual data points, and error bars represent the SEM of the total number of spindles analyzed (N). (C) Caco-2 was transfected with control or Cdc42 siRNA and was fixed and stained for DNA (blue), tubulin (green), and filamentous actin (F-actin; red). Single confocal sections through the center of the cysts are shown. Three z sections are shown for the Cdc42 siRNA cyst to reveal both poles of the misoriented spindle. (D and E) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue) and aPKC (green). (D) Cdc42 siRNA structures contain cells in the middle, with apical domains present between inner and outer cells. (E) Cdc42 siRNA structures possess apical domains that are not in the center and single cells with more than one apical domain. Arrows indicate cells with multiple distinct apical patches on their surface. Bars, 10 μm.

    Article Snippet: RNAi To deplete Cdc42, a SMARTpool (a mixture of four siRNA duplexes) and individual siRNA duplexes were purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Staining

    Cdc42 depletion induces multiple lumens. (A) Caco-2 was transfected with control or Cdc42 siRNA, plated in three dimensions, and treated with CTX at day 6 to induce luminal swelling. Phase images from cysts before (0 h) and after (12 h) treatment are shown. Note that about half of the Cdc42-depleted cysts lack a single central lumen. Bars, 50 μm. (B) Western blot of Caco-2 transfected with control siRNA, Cdc42 siRNA SMARTpool, or two different individual duplexes. Cdc42 levels are significantly reduced by 3 d and remain reduced for 7 d. (C) Caco-2 cultured as in A was fixed and stained with rhodamine phalloidin. Cysts were examined for single lumen (blue) or multiple lumens (red). The mean ± standard deviation for three independent experiments (at least 50 cysts each) is shown. (D and E) Caco-2 cultured as in A was fixed and stained for DNA (blue) and aPKC (green; D) or DNA (blue), E-cadherin (Ecad; green), and ZO-1 (red; E). Single confocal sections through the middle of the cysts are shown. DIC, differential interference contrast. Bars, 25 μm.

    Journal: The Journal of Cell Biology

    Article Title: Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis

    doi: 10.1083/jcb.200807121

    Figure Lengend Snippet: Cdc42 depletion induces multiple lumens. (A) Caco-2 was transfected with control or Cdc42 siRNA, plated in three dimensions, and treated with CTX at day 6 to induce luminal swelling. Phase images from cysts before (0 h) and after (12 h) treatment are shown. Note that about half of the Cdc42-depleted cysts lack a single central lumen. Bars, 50 μm. (B) Western blot of Caco-2 transfected with control siRNA, Cdc42 siRNA SMARTpool, or two different individual duplexes. Cdc42 levels are significantly reduced by 3 d and remain reduced for 7 d. (C) Caco-2 cultured as in A was fixed and stained with rhodamine phalloidin. Cysts were examined for single lumen (blue) or multiple lumens (red). The mean ± standard deviation for three independent experiments (at least 50 cysts each) is shown. (D and E) Caco-2 cultured as in A was fixed and stained for DNA (blue) and aPKC (green; D) or DNA (blue), E-cadherin (Ecad; green), and ZO-1 (red; E). Single confocal sections through the middle of the cysts are shown. DIC, differential interference contrast. Bars, 25 μm.

    Article Snippet: RNAi To deplete Cdc42, a SMARTpool (a mixture of four siRNA duplexes) and individual siRNA duplexes were purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Western Blot, Cell Culture, Staining, Standard Deviation

    Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin RNA (shRNA) sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P

    Journal: Journal of Neuroinflammation

    Article Title: Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling

    doi: 10.1186/1742-2094-9-67

    Figure Lengend Snippet: Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin RNA (shRNA) sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P

    Article Snippet: RNA interference Individual siRNA duplexes were purchased from Dharmacon Inc (Chicago, IL, USA) and tested for knockdown of RNF11 using quantitative RT-PCR (qRT-PCR) in SH-SY5Y cells (not shown).

    Techniques: Luciferase, Activity Assay, Generated, shRNA, Sequencing, Quantitative RT-PCR

    Prolonged nuclear p65 translocation after TNF-α stimulation observed in neuronal cells with knockdown of RNF11. (A) SH-SY5Y cell lines (untransduced, small hairpin RNA (shRNA)-RNF11, shRNA-Scramble) were stimulated with TNF-α for 0, 30, 60 or 120 minutes. Cells were immunostained for p65 (red) and nuclear DNA (Hoechst 333258, blue). Representative images obtained at 0, 30 and 120 minutes after stimulation are shown. (B) Cells were analyzed for overlap of p65-positive and Hoechst 333258-positive pixels. The fold change in percentage overlap of p65- and Hoechst 333258-positive pixels was calculated relative to unstimulated conditions. (C) Murine primary cortical neurons were treated with cytosine arabinoside and transduced with lentiviruses containing shRNA targeted against RNF11 (shRNA-RNF11 neurons) or scramble shRNA sequence (shRNA-Scramble neurons). Quantitative RT-PCR (qRT-PCR) was used to measure relative RNF11 mRNA levels. (D) shRNA-RNF11 or shRNA-Scramble neurons were stimulated for 0, 30 or 120 min before being immunostained for p65 and Hoechst 333258. Transduced cells were analyzed for overlap of p65- and Hoechst 333258-positive cells. The fold change in percentage overlap of p65- and Hoechst 333258-positive pixels was calculated relative to unstimulated conditions. (E) shRNA-Scramble and shRNA-RNF11 cells were stimulated for 0, 30 or 120 minutes. Cytoplasmic and nuclear fractions were resolved by SDS-PAGE. (F) ImageJ software was used to quantify the ratio of the densitometry of the p65 bands in the nuclear fractions to the density of histone 1 immunoreactivity. The ratio for each time point was compared relative to the steady-state ratio, which was set at 100%. (G) ImageJ software was used to quantify the ratio of the densitometry of the p65 bands in the cytoplasmic fractions in a manner similar to that described in (F). All p65 colocalization values are means ± SEM of triplicate experiments. qRT-PCR values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. Western blots of cytoplasmic and nuclear fractions were run from triplicate experiments. ** P

    Journal: Journal of Neuroinflammation

    Article Title: Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling

    doi: 10.1186/1742-2094-9-67

    Figure Lengend Snippet: Prolonged nuclear p65 translocation after TNF-α stimulation observed in neuronal cells with knockdown of RNF11. (A) SH-SY5Y cell lines (untransduced, small hairpin RNA (shRNA)-RNF11, shRNA-Scramble) were stimulated with TNF-α for 0, 30, 60 or 120 minutes. Cells were immunostained for p65 (red) and nuclear DNA (Hoechst 333258, blue). Representative images obtained at 0, 30 and 120 minutes after stimulation are shown. (B) Cells were analyzed for overlap of p65-positive and Hoechst 333258-positive pixels. The fold change in percentage overlap of p65- and Hoechst 333258-positive pixels was calculated relative to unstimulated conditions. (C) Murine primary cortical neurons were treated with cytosine arabinoside and transduced with lentiviruses containing shRNA targeted against RNF11 (shRNA-RNF11 neurons) or scramble shRNA sequence (shRNA-Scramble neurons). Quantitative RT-PCR (qRT-PCR) was used to measure relative RNF11 mRNA levels. (D) shRNA-RNF11 or shRNA-Scramble neurons were stimulated for 0, 30 or 120 min before being immunostained for p65 and Hoechst 333258. Transduced cells were analyzed for overlap of p65- and Hoechst 333258-positive cells. The fold change in percentage overlap of p65- and Hoechst 333258-positive pixels was calculated relative to unstimulated conditions. (E) shRNA-Scramble and shRNA-RNF11 cells were stimulated for 0, 30 or 120 minutes. Cytoplasmic and nuclear fractions were resolved by SDS-PAGE. (F) ImageJ software was used to quantify the ratio of the densitometry of the p65 bands in the nuclear fractions to the density of histone 1 immunoreactivity. The ratio for each time point was compared relative to the steady-state ratio, which was set at 100%. (G) ImageJ software was used to quantify the ratio of the densitometry of the p65 bands in the cytoplasmic fractions in a manner similar to that described in (F). All p65 colocalization values are means ± SEM of triplicate experiments. qRT-PCR values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. Western blots of cytoplasmic and nuclear fractions were run from triplicate experiments. ** P

    Article Snippet: RNA interference Individual siRNA duplexes were purchased from Dharmacon Inc (Chicago, IL, USA) and tested for knockdown of RNF11 using quantitative RT-PCR (qRT-PCR) in SH-SY5Y cells (not shown).

    Techniques: Translocation Assay, shRNA, Transduction, Sequencing, Quantitative RT-PCR, SDS Page, Software, Western Blot

    Knockdown of RNF11 exaggerates inflammatory responses following TNF-α treatment. (A) and (B) Stably transduced SH-SY5Y short hairpin RNA (shRNA)-RNF11 and shRNA-Scramble cells were exposed to 0 or 10 ng/ml TNF-α for 24 hours. The relative amount of mRNA expression in stimulated and unstimulated cells was calculated using quantitative RT-PCR (qRT-PCR) and glucuronidase β (GUSB) as an internal control. (C) Stably transduced SH-SY5Y shRNA-RNF11 and shRNA-Scramble cells were exposed to 0 or 10 ng/ml TNF-α for 4 hours before cells were rinsed and fresh media were replaced in the plate. Media were collected after 20 hours and analyzed for TNF-α protein levels using a human TNF-α ELISA. ND, not detectable. (D) Murine primary cultures were harvested, and qRT-PCR was used to measure relative RNF11 mRNA levels. (E) Murine primary cortical neurons were treated with cytosine arabinoside and transduced with shRNA-Scramble or shRNA-RNF11 lentivirus. Cells were exposed to 0 or 10 ng/ml TNF-α for 4 or 24 hours, and mRNA levels were examined by qRT-PCR. (F) Murine primary cortical neurons were transduced with shRNA-Scramble or shRNA-RNF11 lentivirus. Cells were exposed to 0 or 10 ng/ml TNF-α for 4 or 24 hours, and protein levels of monocyte chemoattractant protein 1 (MCP-1) were measured using a mouse MCP-1 ELISA. qRT-PCR values are expressed as mean comparative cycle threshold ± SEM with GUSB (for SH-SY5Y cells) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (for primary cultures) levels as an internal control for three independent experiments. ELISA values are expressed as averages of protein levels extrapolated from standard curve analysis from three independent experiments. *** P

    Journal: Journal of Neuroinflammation

    Article Title: Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling

    doi: 10.1186/1742-2094-9-67

    Figure Lengend Snippet: Knockdown of RNF11 exaggerates inflammatory responses following TNF-α treatment. (A) and (B) Stably transduced SH-SY5Y short hairpin RNA (shRNA)-RNF11 and shRNA-Scramble cells were exposed to 0 or 10 ng/ml TNF-α for 24 hours. The relative amount of mRNA expression in stimulated and unstimulated cells was calculated using quantitative RT-PCR (qRT-PCR) and glucuronidase β (GUSB) as an internal control. (C) Stably transduced SH-SY5Y shRNA-RNF11 and shRNA-Scramble cells were exposed to 0 or 10 ng/ml TNF-α for 4 hours before cells were rinsed and fresh media were replaced in the plate. Media were collected after 20 hours and analyzed for TNF-α protein levels using a human TNF-α ELISA. ND, not detectable. (D) Murine primary cultures were harvested, and qRT-PCR was used to measure relative RNF11 mRNA levels. (E) Murine primary cortical neurons were treated with cytosine arabinoside and transduced with shRNA-Scramble or shRNA-RNF11 lentivirus. Cells were exposed to 0 or 10 ng/ml TNF-α for 4 or 24 hours, and mRNA levels were examined by qRT-PCR. (F) Murine primary cortical neurons were transduced with shRNA-Scramble or shRNA-RNF11 lentivirus. Cells were exposed to 0 or 10 ng/ml TNF-α for 4 or 24 hours, and protein levels of monocyte chemoattractant protein 1 (MCP-1) were measured using a mouse MCP-1 ELISA. qRT-PCR values are expressed as mean comparative cycle threshold ± SEM with GUSB (for SH-SY5Y cells) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (for primary cultures) levels as an internal control for three independent experiments. ELISA values are expressed as averages of protein levels extrapolated from standard curve analysis from three independent experiments. *** P

    Article Snippet: RNA interference Individual siRNA duplexes were purchased from Dharmacon Inc (Chicago, IL, USA) and tested for knockdown of RNF11 using quantitative RT-PCR (qRT-PCR) in SH-SY5Y cells (not shown).

    Techniques: Stable Transfection, shRNA, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transduction