(A) GST and GST-TRAM were analyzed by SDS-PAGE and Coomassie staining to demonstrate equal inputs for GST pulldown assays in (B) and (C). (B, C) HEK293T cells were transfected with 8 μg empty vector (EV), Flag-TLR2 or Flag-TRIF (B) or with 4 μg Flag-Mal or Flag-MyD88 (C). After 48 h, lysates were incubated with GST-TRAM or GST alone as indicated, and together with input lysates, were analyzed by SDS-PAGE and immunoblotting. Data are representative of at least three independent experiments * = nonspecific band.