Figure 1. TNF induces M1‐linked ubiquitination at mitochondria.

• A, B
  A fraction of OTULIN is localized at mitochondria where it suppresses M1‐linked ubiquitination. HEK293T cells were transfected with control or OTULIN siRNA. Cells were harvested 72 h (A) or 48 h (B) after transfection or 15 min after TNF treatment (25 ng/ml). Mitochondria were isolated by differential centrifugation and purified by ultracentrifugation using an OptiPrep™ density gradient. 38% (A) or 20% (B) of the mitochondrial fractions and 2% (A, B) of whole cell lysates were analyzed by immunoblotting using the antibodies indicated. For the detection of M1‐linked ubiquitin chains, either the 1E3 (A) or the 1F11/3F5/Y102L (B) antibody was used.
• C
  TNF induces M1‐ubiquitination at mitochondria in various cell types. The indicated cell types were treated with TNF (25 ng/ml, 15 min) and purified mitochondrial fractions analyzed as described in (A).
• D
  TNF‐induced mitochondrial M1‐ubiquitination does not occur in HOIP‐deficient cells. Wildtype (WT) and HOIP‐KO HAP cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in (A).
• E
  Mitochondria and M1‐linked ubiquitin co‐localize after TNF treatment. SH‐SY5Y cells were treated with TNF (25 ng/ml, 15 min), fixed, stained with antibodies against HSP60 (green) and M1‐ubiquitin (1E3, red), and analyzed by SR‐SIM. Scale bar, 5 μm.
• F
  TNF induces recruitment of LUBAC components to mitochondria. HEK293T cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in (A).
• G
  TNF induces a fast and transient increase in M1‐ and K63‐specific ubiquitination at mitochondria. HEK 293T cells were treated with TNF (25 ng/ml) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1‐, K63‐, and K48‐specific ubiquitin antibodies.

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