Figure 2. Tankyrase is K63‐linked polyubiquitinated by the E3 ligase RNF8.

• A
  K63‐Ub is enriched in the nuclear fraction. Immunoblot analysis of nuclear and cytosolic extracts with the indicated antibodies.
• B
  K63‐Ub TNKS1 is in the nuclear fraction. Cytosolic and nuclear extracts from HTC75 cells cotransfected with FlagTNKS1 and HA‐K63‐Ub were immunoprecipitated with anti‐Flag antibody and immunoblotted with anti‐Flag or HA antibodies.
• C
  Endogenous RNF8 is bound to endogenous K63‐linked polyubiquitinated tankyrase 1. HTC75 cell extracts were immunoprecipitated with anti‐RNF8 antibody and immunoblotted with anti‐RNF8, TNKS1, or K63‐Ub antibodies.
• D, E
  K63‐Ub chains are diminished on tankyrase 1 in RNF8‐depleted cells. (D) Immunoblot analysis of HTC75 cell lines stably expressing GFP or RNF8 shRNA. (E) GFP or RNF8 shRNA cell lines were cotransfected with FlagTNKS1 and HA‐K63‐Ub, immunoprecipitated with anti‐Flag antibody, and immunoblotted with anti‐Flag or HA antibodies. Protein levels relative to FlagTNKS1 and normalized to the GFP control are indicated below the blot.
• F
  FlagTNKS2 is modified by K63‐Ub chains that are decreased in RNF8‐depleted cells. GFP or RNF8 HTC75 shRNA cell lines were cotransfected with FlagTNKS2 and HA‐K63‐Ub, immunoprecipitated with anti‐Flag, and immunoblotted with anti‐Flag or HA antibodies.
• G
  The decrease in K63‐Ub chains in RNF8‐depleted cells is rescued by RNF8 WT, but not by the C403S RING‐deficient mutant. GFP or RNF8 HTC75 shRNA cell lines were cotransfected with FlagTNKS1, HA‐K63‐Ub, and a vector control (V) or MycRNF8 WT or C403S, immunoprecipitated with anti‐Flag, and immunoblotted with anti‐HA, Flag, or Myc antibodies.
• H
  K63‐Ub chains are diminished on tankyrase 1 in Ubc13‐depleted cells. HTC75 cells were transfected with GFP or Ubc13 siRNA for 24 h, followed by cotransfection with FlagTNKS1 and HA‐K63‐Ub for an additional 24 h. Cell extracts were immunoprecipitated with anti‐Flag and immunoblotted with anti‐HA and anti‐Flag antibodies.
• I
  RNF8 ubiquitinates tankyrase 1 in vitro. Ubiquitination reactions containing the indicated purified proteins were fractionated on SDS–PAGE, visualized by staining with amido black (right panel) and by immunoblotting with anti‐ubiquitin antibody (left panel).
• J
  RNF8 K63‐ubiquitinates tankyrase 1 (but not an unrelated protein GMD) in vitro. Ubiquitination reactions containing the E1, E2, E3, and K63‐Ub and GMD or TNKS1 were fractionated on SDS–PAGE, visualized by staining with amido black (right panel) and by immunoblotting with anti‐ubiquitin antibody (left panel). X indicates a blank lane.

Source data are available online for this figure.