Complementation of IRAK3 Knockout in THP-1 Cells

Complementation of the THP-1 IRAK3^−/− knockout cell line (K6-3) with an empty pcDNA6.2/C-EmGFP-DEST plasmid (control) or with the plasmid encoding eGFP-tagged wild type IRAK3 or selected mutated versions of the protein (R97A, D377A, D385A, R97A/R372L) was performed as described previously [27 (link)]. Transiently transfected IRAK3 knockout cells were treated with 1 µg mL⁻¹ LPS with or without 0.1 nM 8-Br-cGMP for 24 h. Levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured using BD OptEIA IL-6 and OptEIA TNF-α ELISA kits (BD Biosciences) according to the manufacturer’s instructions. Data from 7–9 independent experiments were normalized to the eGFP fluorescence signal (excitation: 470–15 nm, emission: 515–20 nm) measured by CLARIOstar Plus and total protein concentration quantified by the Bradford method [58 (link)] and analyzed (p value < 0.05; one-way ANOVA followed by Turkey’s multiple comparisons test).