Fig 1: TBK1 phosphorylates ATAD3A at Ser321 to promote cellular senescence. A) Immunoblot analysis of the interaction between endogenous ATAD3A and TBK1 in normal or senescent A549 and WI‐38 cells (n = 3). B) Confocal imaging of ATAD3A (green) and TBK1 (red) co‐localization in normal or senescent A549 and WI‐38 cells (n = 3). Areas outlined by squares in the merged images are enlarged at right. Scale bars, 5 µm. C) Immunoblot analysis of phosphorylated ATAD3A in senescent cells using a p‐Ser‐ and p‐Thr‐specific antibody (n = 3). D) In vitro TBK1 kinase assay using recombinant TBK1 and Flag‐ATAD3A purified from 293T cells as the substrate (n = 3). E) Listing of 2 ATAD3A Ser phosphorylation sites (highlighted in red) identified via MS analysis (n = 2). F) Immunoblot analyzing the phosphorylation of transfected Flag‐ATAD3A or Ser‐mutated Flag‐ATAD3A (n = 3). G) In vitro TBK1 kinase assay using recombinant TBK1 and Flag‐ATAD3A, Flag‐ATAD3A S321A purified from stable A549 cell lines as the substrate (n = 3). H) Immunoblot analysis of TBK1 in stable negative control (shNC) or shTBK1‐expressing A549 cells (n = 3). I) SA‐β‐gal staining of shNC and shTBK1 A549 cells. Cells were initially induced with doxorubicin (150 nm) for 48 h, followed by additional culturing for 48 h. Scale bars, 100 µm. J) SA‐β‐gal staining of NC and ATAD3A KO A549 cells stably expressing Flag‐ATAD3A, Flag‐ATAD3A S321A, or Flag‐ATAD3A S321E (n = 3). Cells were initially induced with doxorubicin (150 nm) for 48 h, followed by additional culturing for 48 h. Scale bars, 100 µm. K) SA‐β‐gal staining of WI‐38 and MSC cells transfected with Flag‐ATAD3A, Flag‐ATAD3A S321A or Flag‐ATAD3A S321E (n = 3). Scale bars, 100 µm. L) SA‐β‐gal staining of shNC, shTBK1, and shNC or shTBK1 plus simultaneously expressed Flag‐ATAD3A or Flag‐ATAD3A S321E A549 cells (n = 3). Cells were initially induced with doxorubicin (150 nm) for 48 h, followed by additional culturing for 48 h. Scale bars, 100 µm.
Fig 2: Increased activation and mitochondrial localization of TBK1 in senescent cells. A) SA‐β‐gal staining of normal and senescent cells in various senescent cell models (n = 3). P: passages, Dox: Doxorubicin (150 nm), WT: wild type, HGPS: Hutchinson–Gilford progeria syndrome. Scale bars, 50 µm. B) Immunoblot analysis of phosphorylated TBK1 (pS172‐TBK1) and TBK1 in normal or senescent cells (n = 3). C) Confocal imaging of TBK1 (red) and Tom20 (green) co‐localization in normal or senescent cells (n = 3). Areas outlined by squares in the merged images are enlarged at right. Scale bars, 5 µm. D) Transmission electron microscopy (TEM) images of TBK1 labeled by antibody‐conjugated gold particles in normal or senescent cells (n = 3). m: mitochondria. Scale bars, 100 nm. E) Immunoblot analysis of p‐TBK1 and TBK1 in the mitochondrial fraction of normal or senescent cells (n = 3). Tom40: mitochondria marker, GAPDH: whole cell marker.
Fig 3: TBK1‐ATAD3A‐Pink1 axis inhibits mitophagy and promotes cellular senescence. A) SA‐β‐gal staining of indicated A549 cells (n = 3). Cells were initially induced with DMSO or doxorubicin (150 nm) for 48 h, followed by additional culturing for 48 h. B) Immunoblot analysis of p53, p21, and Pink1 in the cells described in A (n = 3). C) Confocal imaging of Tom20 (green) and LAMP1 (red) in indicated cells (n = 3). Cells were initially induced with doxorubicin (150 nm) for 48 h, followed by additional culturing for 48 h. Areas outlined by squares in the merged images are enlarged at right. Scale bars, 2 µm. D) SA‐β‐gal staining of NC, shTBK1, shPink1, and cells with combined shPink1 and shTBK1 (n = 3). The indicated A549 cells were initially induced with DMSO or doxorubicin (150 nm) for 48 h, followed by additional culturing for 48 h. E) Immunoblot analysis of p53, p21, and Pink1 in the cells described in D (n = 3). F) Confocal imaging of Tom20 (green) and LAMP1 (red) in NC, shTBK1, shPink1, and cells with combined shPink1 and shTBK1 (n = 3). Cells were initially induced with doxorubicin (150 nm) for 48 h, followed by additional culturing for 48 h. Areas outlined by squares in the merged images are enlarged at right. Scale bars, 2 µm.
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