Fig 6.

ASB3 specifically interacts with TRAF6. (a) HEK293T cells were transfected with HA-ASB3 plasmid or a vector control. Twenty-four hours after transfection, the cells were treated with TNF-α (100 ng/mL) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (b) HEK293T cells were transfected with the indicated plasmids along with control vector or increased amounts of ASB3 expression plasmids. Reporter assays were performed 24 h after transfection. (c) HEK293T cells were cotransfected with HA-ASB3 and Flag-TRAF6 plasmids or a vector control. (d and f) The indicated plasmids were transfected into HEK293T cells or transcribed in vitro. Then, coimmunoprecipitation (Co-IP) and immunoblotting analyses were performed with the indicated antibodies. (e) HEK293T cells were transfected with HA-ASB3, Flag-TRAF6, or Flag-TRAF3 plasmids for 24 h and then stained with Flag antibody or HA primary antibody and secondary antibody. The nuclei were stained with DAPI. The fluorescence intensity profile of DAPI (blue), HA-ASB3 (green), and Flag-TRAF6 or Flag-TRAF3 (red) was measured along the line drawn by ZEN Blue. Scale bars, 50 µm. (g and h) Semi-endogenous and endogenous Co-IP analysis of the interaction of ASB3 with TRAF6 in HEK293T or HT-29 cells treated with TNF-α for 24 h (100 ng/mL). (i and j) Co-IP analysis of the interaction of TRAF6 with ASB3 or its truncation mutants in HEK293T cells. Differences were determined in panel b by unpaired Student’s t test.