Stable THP-1 cells expressing shRNA
The lentiviral plasmid pLKO.1 expressing shRNA targeting human MyD88 was purchased from Open Biosystems (www.openbiosystems.com) and was validated in our laboratory. shRNA targeting human IRAK1 (forward primer 5′-CCGGAGCAGCTGTCCAGGTTTCGTCTCATAAAACCTGGACAGCTGCTCCTTTTTG-3′, reverse primer 5′-AATTCAAAAAGGAGCAGCTGTCCAGGTTTTATGAGACGAAACCTGGACAGCTGCT-3′ mRNA (IRAK1 mRNA target sequence is underlined) and human TRAF6 (forward primer 5′-CCGGAGAAACCTGTTGTGATTCGTCTCATAAATCACAACAGGTTTCTCCTTTTTG-3′, reverse primer 5′-AATTCAAAAAGGAGAAACCTGTTGTGATTTATGAGACGAATCACAACAGGTTTCT-3′ (TRAF6 mRNA target sequence is underlined) were designed in our laboratory and were cloned into the pLKO.1 plasmid. Lentiviruses encoding shRNA sequences were generated by transfecting the packaging cell line HEK-293T with the shRNA-encoding pLKO.1 plasmids in combination with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G using Effectene transfection reagent (Qiagen, www.qiagen.com). Supernatants were collected 48 hours post-transfection, clarified by centrifugation, and stored at −80°C. THP-1 cells were transduced with the lentiviral particles by culturing the cells with supernatants from the virus-producing cells in the presence of 8 µg/ml polybrene (Millipore, www.millipore.com) and spinoculation for two hours at 2000 RPM. Successfully transduced cells were selected and expanded by treatment with 0.8 µg/ml puromycin.