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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
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1) Product Images from "Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling"

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

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-9-67

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

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

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Quantitative RT-PCR:

Article Title: Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling
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). .. The most effective sequence was cloned into pFH1UGW backbone by introducing Nhe1 and Pac1 overhangs at each end of the duplex.

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