Transfection and Co-immunoprecipitation Assays

MyD88-deficient HEK293-I3A and Huh7 cells were transiently transfected using Lipofectamine^TM 2000 (Invitrogen, Burlington, ON, Canada) as recommended by the manufacturer with indicated plasmids. The total amount of DNA was kept constant. For assessment of SMAD4 and MyD88 interactions, MyD88 KO HEK293-I3A cells were used. Twenty-four hours after transfection, cells were lysed in 1 mL RIPA buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, and protease inhibitor cocktail (Complete Mini, Roche, Mannheim, Germany). Cell lysates were then incubated with indicated antibodies (anti-Flag, anti-HA, or anti-MyD88 antibody) for 3 h at 4°C, after which EZview Red Protein A Affinity Gel (Sigma-Aldrich) was added for another 2 h. For control reactions, mouse IgG1 (Santa Cruz) was used. The immune complexes were precipitated and washed thoroughly with RIPA buffer. Immunoprecipitated proteins were then eluted by adding sample buffer and were subsequently fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by immunoblotting with anti-Flag and anti-HA antibodies. Lysates were also immunoblotted for expression of transfected SMAD4-Flag (pRK-DPC4-Flag) and HA-MyD88 (pCMV-HA-MyD88).