Figure 3. K63‐Ub chains increase tankyrase 1 stability.

• A
  Endogenous tankyrase 1 is modified and stabilized by K63‐Ub chains. HTC75 cells were transfected with vector (V) or HA‐K63‐Ub, immunoprecipitated with anti‐HA, and immunoblotted with anti‐HA or TNKS1 antibodies.
• B, C
  Neither HA‐K48‐Ub nor HA‐K63R‐Ub induced stabilization of tankyrase 1. HTC75 cells were transfected with V, HA‐K63‐Ub, and (B) HA‐K48‐Ub or (C) HA‐K63R‐Ub and analyzed by immunoblot with anti‐TNKS1 or α‐tubulin antibodies.
• D, E
  HA‐K63‐Ub TNKS1 has a longer half‐life than HA‐K48‐Ub TNKS1. (D) HTC75 cells were cotransfected with FlagTNKS1 and HA‐K63‐Ub or HA‐K48‐Ub, treated with cycloheximide for 0, 3, or 6 h prior to harvest, immunoprecipitated with anti‐Flag, and immunoblotted with anti‐Flag or HA antibodies. Protein levels of HA‐K63‐Ub or HA‐K48‐Ub TNKS1 relative to FlagTNKS1 and normalized to the 0‐h time point are indicated below the blot. (E) Graphical representation of the fold change. Average of two independent experiments ± SEM. *P ≤ 0.05; Student's unpaired t‐test.
• F, G
  The half‐life of nuclear tankyrase 1 is decreased in RNF8‐depleted cells. (F) Nuclear extracts were prepared from GFP or RNF8 shRNA HTC75 cells following treatment with cycloheximide for 0, 3, or 6 h and analyzed by immunoblotting with anti‐TNKS1 or lamin B antibodies. Protein levels of tankyrase 1 in GFP shRNA‐ or RNF8 shRNA‐depleted cells relative to lamin B and normalized to the 0‐h time point are indicated below the blot. (G) Graphical representation of the fold change. Average of two independent experiments ± SEM. *P ≤ 0.05; Student's unpaired t‐test.
• H
  Tankyrase 1 protein levels are decreased in nuclear extracts from RNF8‐depleted cells. Graphical representation of the fold change in tankyrase 1 relative to lamin B measured by immunoblot. Average of four independent experiments ± SD. **P ≤ 0.01; Student's unpaired t‐test.
• I
  RNF8 protects tankyrase 1 from proteasomal degradation. Immunoblot analysis of nuclear extracts from HTC75 cell lines stably expressing RNF8 or GFP shRNA treated with and without MG132 for 4 h prior to harvest.

Source data are available online for this figure.