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Horizon Discovery rnf11 sirna duplex
<t>RNF11</t> is essential for the termination of NF-κB signalling. ( A ) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK and RNF11. ( B ) THP-1 cells were transfected on consecutive days with control scrambled, RNF11 or A20 siRNAs. At 2 days after the second transfection, cells were treated with LPS (1 μg/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK, A20, RNF11 and β-actin. ( C ) THP-1 cells were transfected with control scrambled or RNF11 <t>siRNA</t> as described in (A). At 2 days after the second transfection, cells were stimulated with TNF (10 ng/ml) for various times and RNA was subjected to real-time PCR for IκBα and A20 expression. This experiment was repeated twice with similar results. ( D ) THP-1 cells were transfected with siRNAs as described in (A). Supernatants were subjected to an IL-6 ELISA. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way ANOVA, followed by the Tukey–Kramer test for multiple comparisons. * P
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1) Product Images from "The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling"

Article Title: The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling

Journal: The EMBO Journal

doi: 10.1038/emboj.2008.285

RNF11 is essential for the termination of NF-κB signalling. ( A ) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK and RNF11. ( B ) THP-1 cells were transfected on consecutive days with control scrambled, RNF11 or A20 siRNAs. At 2 days after the second transfection, cells were treated with LPS (1 μg/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK, A20, RNF11 and β-actin. ( C ) THP-1 cells were transfected with control scrambled or RNF11 siRNA as described in (A). At 2 days after the second transfection, cells were stimulated with TNF (10 ng/ml) for various times and RNA was subjected to real-time PCR for IκBα and A20 expression. This experiment was repeated twice with similar results. ( D ) THP-1 cells were transfected with siRNAs as described in (A). Supernatants were subjected to an IL-6 ELISA. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way ANOVA, followed by the Tukey–Kramer test for multiple comparisons. * P
Figure Legend Snippet: RNF11 is essential for the termination of NF-κB signalling. ( A ) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK and RNF11. ( B ) THP-1 cells were transfected on consecutive days with control scrambled, RNF11 or A20 siRNAs. At 2 days after the second transfection, cells were treated with LPS (1 μg/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK, A20, RNF11 and β-actin. ( C ) THP-1 cells were transfected with control scrambled or RNF11 siRNA as described in (A). At 2 days after the second transfection, cells were stimulated with TNF (10 ng/ml) for various times and RNA was subjected to real-time PCR for IκBα and A20 expression. This experiment was repeated twice with similar results. ( D ) THP-1 cells were transfected with siRNAs as described in (A). Supernatants were subjected to an IL-6 ELISA. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way ANOVA, followed by the Tukey–Kramer test for multiple comparisons. * P

Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

RNF11 specifically inhibits NF-κB activation. ( A ) THP-1 cells were transfected on day 1 with no siRNA (mock), control scrambled siRNA or RNF11 siRNA. On day 2, the same cells were transfected with pRL-tk internal control Renilla luciferase plasmid, κB-TATA Luc and Myc–RNF11 as indicated. After an additional 2 days, cells were treated with TNF (10 ng/ml) for 8 h and lysates were subjected to dual luciferase assays. The lysates were also subjected to immunoblotting to examine the expression of RNF11 and β-actin. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by the Tukey–Kramer test for multiple comparisons ( * P
Figure Legend Snippet: RNF11 specifically inhibits NF-κB activation. ( A ) THP-1 cells were transfected on day 1 with no siRNA (mock), control scrambled siRNA or RNF11 siRNA. On day 2, the same cells were transfected with pRL-tk internal control Renilla luciferase plasmid, κB-TATA Luc and Myc–RNF11 as indicated. After an additional 2 days, cells were treated with TNF (10 ng/ml) for 8 h and lysates were subjected to dual luciferase assays. The lysates were also subjected to immunoblotting to examine the expression of RNF11 and β-actin. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by the Tukey–Kramer test for multiple comparisons ( * P

Techniques Used: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing

RNF11 requires the RING domain and PPXY motif to interact with the A20 ubiquitin-editing complex and to terminate NF-κB signalling. ( A ) Schematic diagram of RNF11 domain organization and mutants used in this figure. The PPXY motif is located between amino acids 37 and 40. The RING finger domain is located between amino acids 99 and 140. The RNF11 C99A mutant has a non-functional RING domain. The RNF11 Y40A mutant has a disrupted PPXY motif. ( B ) THP-1 cells were transfected with a control scrambled or a single duplex RNF11 siRNA. The following day, the cells were transfected with siRNA-resistant forms of Flag–RNF11 (WT R ), Flag–RNF11 C99A (C99A R ) and Flag–RNF11 Y40A (Y40A R ). After two more days, cells were treated with TNF (10 ng/ml) for the indicated times and lysates were immunoblotted with anti-pIκBα, IκBα, pJNK, JNK, Flag, RNF11 and β-actin. ( C ) THP-1 cells were transfected with siRNA and siRNA-resistant forms of RNF11 as described in (B). At 2 days after DNA transfections, cells were treated with TNF for 30 min where indicated. Lysates were subjected to immunoprecipitations with anti-RNF11 followed by immunoblotting with A20, TAX1BP1 and RIP1. The lysates were also used for immunoblotting using anti-IκBα, Flag, RNF11 and β-actin.
Figure Legend Snippet: RNF11 requires the RING domain and PPXY motif to interact with the A20 ubiquitin-editing complex and to terminate NF-κB signalling. ( A ) Schematic diagram of RNF11 domain organization and mutants used in this figure. The PPXY motif is located between amino acids 37 and 40. The RING finger domain is located between amino acids 99 and 140. The RNF11 C99A mutant has a non-functional RING domain. The RNF11 Y40A mutant has a disrupted PPXY motif. ( B ) THP-1 cells were transfected with a control scrambled or a single duplex RNF11 siRNA. The following day, the cells were transfected with siRNA-resistant forms of Flag–RNF11 (WT R ), Flag–RNF11 C99A (C99A R ) and Flag–RNF11 Y40A (Y40A R ). After two more days, cells were treated with TNF (10 ng/ml) for the indicated times and lysates were immunoblotted with anti-pIκBα, IκBα, pJNK, JNK, Flag, RNF11 and β-actin. ( C ) THP-1 cells were transfected with siRNA and siRNA-resistant forms of RNF11 as described in (B). At 2 days after DNA transfections, cells were treated with TNF for 30 min where indicated. Lysates were subjected to immunoprecipitations with anti-RNF11 followed by immunoblotting with A20, TAX1BP1 and RIP1. The lysates were also used for immunoblotting using anti-IκBα, Flag, RNF11 and β-actin.

Techniques Used: Mutagenesis, Functional Assay, Transfection