For ILEI and mammalian non-targeting control shRNA knock-down in NCI-H1993, NCI-H441, MKN45, OE33 and SKBR3 cells, MISSION shRNA lentiviral transduction particles (Sigma, St Louis, MS, USA) were used according to the manufacturer’s instructions. Five shRNA sequences were pretested for ILEI knock-down (sh261 CCGGGATGCAAGTTTAGGAAATCTACTCGAGTAGATTTCCTAAACTTGCATCTTTTTG, sh328 CCGGCCAGATATAAGTGTGGGATCTCTCGAGAGATCCCACACTTATATCTGGTTTTTG, sh506 CCGGAGGAGAAGTATTAGACACTAACTCGAGTTAGTGTCTAATACTTCTCCTTTTTTG, sh579 CCGGGCCATACAAGATGGAACAATACTCGAGTATTGTTCCATCTTGTATGGCTTTTTG and sh1767 CCGGCCTGTGTTTATCTAACTTCATCTCGAGATGAAGTTAGATAAACACAGGTTTTTG) and two were selected (sh261 and sh506) as the most efficient for later studies. In studies with only one shILEI cell line, “shILEI” indicates sh506. Stable cell lines were established using selection for puromycin resistance of transduced cells. ILEI expression was validated in whole cell lysates and conditioned medium (CM) by Western blotting.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.