Author: Yen, Wei-Chen; Wu, Yi-Hsuan; Wu, Chih-Ching; Lin, Hsin-Ru; Stern, Arnold; Chen, Shih-Hsiang; Shu, Jwu-Ching; Tsun-Yee Chiu, Daniel
Title: Impaired inflammasome activation and bacterial clearance in G6PD deficiency due to defective NOX/p38 MAPK/AP-1 redox signaling Document date: 2019_11_2
ID: 6fw4thkq_26
Snippet: The human IL-1β promoter plasmid was a kind gift from Dr. Ben-Kuen Chen (National Cheng Kung University) [40] . The deletion sequence of the AP-1 binding site bearing the luciferase plasmid was constructed using site-directed mutagenesis. The 293FT cells cultured in 24-well plates were transfected with 600 ng of pLKO-scramble or pLKO-G6PD [38] together with 300 ng of the IL-1β promoter plasmid (wild-type or deletion) and 100 ng of pRL-TK vector.....
Document: The human IL-1β promoter plasmid was a kind gift from Dr. Ben-Kuen Chen (National Cheng Kung University) [40] . The deletion sequence of the AP-1 binding site bearing the luciferase plasmid was constructed using site-directed mutagenesis. The 293FT cells cultured in 24-well plates were transfected with 600 ng of pLKO-scramble or pLKO-G6PD [38] together with 300 ng of the IL-1β promoter plasmid (wild-type or deletion) and 100 ng of pRL-TK vector by Lipofectamine® 2000 (Thermo Fisher Scientific, MA, USA). Forty-eight hours after transfection, the luciferase activity was measured by using the Dual-Luciferase Assay (Promega, MA, USA) with a GLOMAX luminometer (Promega, MA, USA). The cellular extracts were assayed for luciferase activity and normalized to the Renilla luciferase levels. The data are presented relative to the pLKO-scramble levels (relative light unit; RLU).
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