siRNA ADAM17

siRNA / miRNA gene silencing Human - BOSC23 ADAM17

Experiment
siRNA / miRNA gene silencing Human - BOSC23 ADAM17
Product
siRNA ADAM17 from Dharmacon
Manufacturer
Dharmacon

Protocol tips

Publication protocol

RNAi was used to specifically knock down ADAM17 (AAI46659) expression in Bosc23 cells. Small interfering RNAs (siRNAs) were expressed from short hairpin RNAs (shRNAs) using the MISSION™ shRNA (Sigma) plasmids TRCN0000052168 to TRCN0000052172. Cells were co-transfected with plasmid encoding ShhNp cDNA and shRNA plasmids or empty control plasmid, and transfected cells were enriched by the addition of 5 μg/ml puromycin for 30 h before starting the assay. In addition, to confirm findings obtained by shRNAi, ADAM17 was knocked down in ShhNp-expressing Bosc23 cells by using 5 nmol of On-Target plus anti-human ADAM17 pooled siRNA (Dharmacon) using the siGENOME nontargeting siRNA pool as a control. In all experiments, the amount of ShhNp released into the medium was quantified using ImageJ. Data were plotted as released versus cell-bound ShhNp relative to the value obtained with the empty vector control, which was set to 100%. Statistical analysis was performed in Excel using Student's t test (two-tailed, unpaired). All values shown throughout are ±S.D. To assess the effectiveness of ADAM knockdown, semiquantitative RT-PCR analysis of ADAM17 and glypican 1 (Glp1) expression in Bosc23 cells transfected with pooled siRNAs specifically interfering with ADAM17 mRNA was conducted. PCR products were quantified using ImageJ, and their relative intensities were calculated. Semiquantitative RT-PCR revealed a 60% reduction in ADAM17 mRNA levels in Bosc23 cells cotransfected with five pooled shRNAs specific for the protease. As a control, glypican 1 mRNA levels were not affected by ADAM17-specific mRNA knockdown.

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Papers

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Paper title
Heparan Sulfate-modulated, Metalloprotease-mediated Sonic Hedgehog Release from Producing Cells
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Manufacturer protocol

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