60 mm culture plate

Cells were cultured at a density of 100 × 10⁴ cells in a 60-mm culture plate (Tarsons-960020). Four hours before the addition of the TLR3 ligand, the MyD88 inhibitor ST2825 was added and incubated for 24 h. Cells were lysed with non-denaturing lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA) with a protease inhibitor cocktail. The lysate was incubated on ice for 30 min and centrifuged at 10,000 rpm for 20 min at 4°C. The supernatant was incubated with 1 μg of an indicated antibody and Dynabeads (Invitrogen-10003D) overnight at 4°C. The Dynabeads were pelleted down and washed with lysis buffer after overnight incubation. The precipitates were resolved in SDS-PAGE and subjected to Western blotting with the indicated antibodies.

Cells were cultured at a density of 100 × 10⁴ cells in a 60-mm culture plate (Tarsons-960020). Four hours before the addition of the TLR3 ligand, the MyD88 inhibitor was added and incubated for 24 h. Cells were lysed using RIPA buffer, and gel electrophoresis was performed using acrylamide gel. Proteins were transferred to PVDF membranes and blotted with antibodies against interleukin 1 receptor-associated kinase 1 (IRAK1; Invitrogen-38-5600), phospho IRAK1–Thr209, (Invitrogen-PA5-38633), transforming growth factor beta-activated kinase 1 (TAK1; Invitrogen-700 113), phospho TAK1–Thr184/187 (Invitrogen-MA5-15073), TGF-beta-activated kinase 1 (TAB1; Invitrogen-PA5-28683), TNF receptor-associated factor 6 (TRAF6; Invitrogen-PA5-29622), and cyclin D1 (Invitrogen-AHF0082). The antibody against β-actin (Invitrogen-MA191399) was used as a loading control.