TBK1 knockdown and rescue in Hela-M cells

TANK-binding kinase 1 (TBK1) is a multifunctional kinase with roles in several crucial cell processes, including innate immune response, anti-viral response, and mitochondrial clearance, the last of which is of most interest to us. In order to further understand the role of TBK1 in mitophagy, we developed a protocol to transiently deplete TBK1 from a model system, HeLa cells, and re-introduce a tagged TBK1 along with other relevant components of mitophagy. Because of the many processes that rely on TBK1, cells depleted of the kinase exhibit poor health. Our protocol accomplishes ~70% depletion of endogenous TBK1 within 24 hours without causing excessive cell death. We employ this protocol to carry out biochemistry experiments such as Western blotting and organelle fractionation, and imaging experiments such as immunofluorescence, and live cell imaging.