Fig. 7

TBK1 mediates the degradation of BinCARD1 through the autophagy pathway. A TBK1-mediated degradation of BinCARD1 was analyzed by western blotting. HEK293T cells were transfected with plasmids expressing Myc-tagged BinCARD1 and gradually increasing amounts of Flag-tagged TBK1 individually or in combination. At 36 h p.t., cell lysate proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-Myc pAb. B, C Western blotting was performed to identify the pathway through which TBK1 degrades BinCARD1. HEK293T cells were transfected with plasmids expressing BinCARD1, and at a 12-hour interval, the cells were transfected with an empty vector or a TBK1-expressing plasmid. Twenty-four hours later, the cells were treated for 8 h with H₂O or 3-MA (B) and DMSO or MG132 (C). Cell lysate proteins were subjected to western blotting with a mouse anti-BinCARD1 mAb or a rabbit anti-TBK1 mAb. D The effect of autophagy inducers on the level of BinCARD1 was assessed. HEK293T cells were transfected with plasmids expressing BinCARD1. At 24 h p.t., cells were treated with rapamycin for 24 h or EBSS for 2 h. Cell lysate proteins were subjected to western blotting with a mouse anti-BinCARD1 mAb