Figure 5. Mitochondria serve as a signaling platform for TNF‐induced NF‐κB activation.

• A
  NF‐κB signaling components are recruited to mitochondria upon TNF treatment. (A) HEK293T cells were treated with TNF (25 ng/ml, 15 min), harvested and purified mitochondrial fractions were analyzed by immunoblotting using the antibodies indicated; p‐p65: phospho‐S536‐p65; p‐TBK1: phospho‐S172‐TBK1.
• B
  Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment. Representative immunofluorescence images of SH‐SY5Y cells using the proximity ligation assay (PLA) between phospho‐S536‐p65 (rabbit) and TOM20 (mouse) coupled antibodies. One set of cells was treated with TNF (25 ng/ml, 15 min) before fixation. Nuclei were stained with DAPI, mitochondria were stained with MitoTracker™ Red CMXRos (red), and the PLA amplification reaction was visualized by green foci. As a positive control, fixed cells were incubated with primary TOM20 (mouse) and TOM70 (rabbit) antibodies and subjected to the PLA assay. As negative controls, fixed cells were incubated with either TOM20 or p‐p65 antibodies prior to the PLA assay. Scale bar, 10 μm.
• C
  Quantification of the phospho‐S536‐p65/TOM20 PLA foci per cell. Data represent the number of PLA foci per cell (mean ± SD); at least 84 cells in total were analyzed per condition. ****P < 0.0001. A two‐tailed nonparametric Mann–Whitney test was used to analyze statistical significance.

Source data are available online for this figure.