Myc-tagged viral Pellino and Pellino3S were cloned into the vector pRSET A-His, expressed in Escherichia coli (BL21 cells) and purified using the His-bind purification kit (Qiagen). For the in vitro ubiquitination assay, recombinant Pellino protein (1 μg) was incubated with ubiquitin (1 μg), E1 (50 ng), UbcH13/Uev1a (400 ng) and protease inhibitor mix (EDTA free) in 5 mM Tris-HCl, pH 7.5, containing MgCl2 (2 mM), ATP (2 mM) and NaCl (100 mM). Reactions were incubated at 37°C for 2 h and terminated by addition of SDS-PAGE sample buffer. Samples were then resolved by SDS-PAGE and analysed by immunoblotting using an anti-ubiquitin antibody

(Santa Cruz). Drosophila Schneider JQ1 2 (S2) cells were cultured in Schneider’s Insect Medium supplemented with 10% v/v fetal bovine serum, penicillin G (100 μg/mL) and streptomycin (100 μg/mL). Cells were maintained at 25°C without CO2 buffering. C-106 stimulation was performed on cells in serum-containing medium at 25°C. HEK293T cells and HEK 293-TLR4 cells (gift from Douglas Golenbock) and U373 cells were cultured in DMEM supplemented with 10% v/v fetal bovine serum, penicillin G (100 μg/mL) and streptomycin (100 μg/mL). G418 (0.5 mg/mL) was used as a selective agent for the stably transfected 293-TLR4 cells.

LPS see more stimulation was performed on cells in serum-containing medium at 37°C. Cells were seeded at 1.8×105 and 2×105/mL, respectively, in 96-well plates (200 μL/well) and 6-well plates (3 mL/well) and grown for 24 h to approximately 80% confluency. Cells were transfected using Lipofectamine (Invitrogen), with each well in a 6-well

and 96-well plate being transfected with 4 μg and 230 ng total Gefitinib in vitro DNA, respectively. For 96-well plate transfections, lysates were generated using Reporter Lysis Buffer (Sigma). Firefly activity and Renilla luciferase activities were assayed using luciferase substrate (Promega) and coelenterazine (0.1 μg/mL in PBS), respectively. Cells were seeded at 2×106/mL in 12-well plates and grown for 24 h. Transfection was then performed using the Calcium Phosphate Transfection kit (Invitrogen) according to the manufacturer’s instructions. For each well, a total of 1 μg DNA was used. In total, 24 h post-transfection cells were washed twice in serum-free Schneider’s Medium, re-seeded in fresh medium and stimulated overnight with or without C-106 ligand. Lysates were generated with Reporter Lysis Buffer (Promega) and assayed for firefly luciferase activity. β-Galactosidase activity was assayed by incubating cell lysate with o-nitrophenyl-β-galactoside (1 mg/mL) at 37°C for 15 min before reading absorbance at 420 nm. Briefly, 24 h post-transfection, cells were lysed in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 0.5% v/v Igepal, 50 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor mixture (25 μg/mL leupeptin, 25 μg/mL aprotinin, 1 mM benzamidine and 10 μg/mL trypsin inhibitor).