A 2000-bp (−2000/+1) and shortcut fragment of the A20 gene promoter was cloned into the pGL3-basic expression vector (herein called p2000, p1300, and p1000) using primers described in table S1. RAW264.7 cells were cultured in 12-well plates and were transfected with these plasmids using an Amaxa Nucleofector (4D-Nucleofector, Lonza, Allendale, NJ, USA). At 48 hours after transfection, cells were further treated with LPS (500 ng/ml) for an additional 12 hours. Thereafter, cells were washed with PBS and lysed in a reporter lysis buffer (Invitrogen). Luciferase reporter activities were measured in triplicate using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol and quantified using the GloMax 96-well plate luminometer (Promega). The firefly luciferase–to–Renilla luciferase ratios were determined and were defined as the relative luciferase activity. Results are shown as the mean ± SEM of a representative experiment performed in triplicate. To examine the transfection efficiency, RAW264.7 cells were transfected with a pmaxGFP control vector, and transfection efficiency was assessed by in situ green fluorescent protein (GFP) expression according to the manufacturer’s protocol (Lonza).

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