Figure 1. Tankyrase 1 is a target of ABRO1 and the BRISC DUB.

• A
  Identification of tankyrase 1 binding proteins by MudPIT MS/MS. Unique peptides/total peptide numbers are indicated.
• B
  Schematic diagram showing the four‐subunit BRISC complex with a line diagram below indicating ABRO1 containing a consensus tankyrase‐binding motif.
• C
  Alignment of the tankyrase‐binding site in ABRO1 across multiple species. Identical amino acids are in black.
• D
  FlagABRO1 WT, but not the tankyrase‐binding site mutant G338R, binds tankyrase 1. Lysates from HTC75 cells transfected with vector (V), FlagABRO1.WT, or FlagABRO1.G338R were immunoprecipitated with anti‐Flag antibody and immunoblotted with anti‐Flag or TNKS1 antibodies.
• E
  Reciprocal coimmunoprecipitation of endogenous tankyrase 1 and BRCC36. HTC75 cell lysates were immunoprecipitated with anti‐TNKS1, BRCC36, or IgG antibodies and immunoblotted with anti‐TNKS1 or BRCC36 antibodies.
• F
  Endogenous tankyrase 1 is K63‐linked polyubiquitinated. HTC75 cell extracts were immunoprecipitated with anti‐K63‐Ub antibody and immunoblotted with antibodies against K63‐Ub or tankyrase 1.
• G
  Endogenous tankyrase 1 is K63‐linked polyubiquitinated in multiple cell lines. Whole‐cell extracts from HTC75 (HT) or MCF7 (MC) cells were immunoprecipitated with anti‐K63‐Ub antibody and immunoblotted with anti‐TNKS1 antibody.
• H
  FlagTNKS1 is K63‐linked polyubiquitinated. HTC75 cells were cotransfected with FlagTNKS1 and HA‐K63‐Ub, immunoprecipitated with anti‐Flag, and immunoblotted with anti‐Flag or HA antibodies.
• I, J
  K63‐Ub chains are increased on tankyrase 1 in BRCC36‐depleted cells. (I) Immunoblot analysis of HTC75 cell lines stably expressing GFP or BRCC36 shRNA. (J) GFP or BRCC36 HTC75 shRNA cell lines were cotransfected with FlagTNKS1 and HA‐K63‐Ub, immunoprecipitated with anti‐Flag, and immunoblotted with anti‐Flag or HA antibodies. Protein levels relative to FlagTNKS1 and normalized to the GFP control are indicated below the blot.
• K, L
  K63‐Ub chains are increased on tankyrase 1 in ABRO1‐depleted cells. (K) Immunoblot analysis of HTC75 cell lines stably expressing GFP or ABRO1 shRNA. (L) GFP or ABRO1 HTC75 shRNA cell lines were cotransfected with FlagTNKS1 and HA‐K63‐Ub, immunoprecipitated with anti‐Flag, and immunoblotted with anti‐Flag or HA antibodies. Protein levels relative to FlagTNKS1 and normalized to the GFP control are indicated below the blot.
• M
  The increase in K63‐Ub chains in ABRO1‐depleted cells is rescued by ABRO1 WT, but not by the G338R tankyrase‐binding mutant. GFP or ABRO1 HTC75 shRNA cell lines were cotransfected with FlagTNKS1, HA‐K63‐Ub, and a vector control or MycABRO1 WT or G338R, immunoprecipitated with anti‐Flag, and immunoblotted with anti‐HA, Flag, or Myc antibodies.
• N
  FlagTNKS2 is modified by K63‐Ub chains that are increased in ABRO1‐depleted cells. GFP or ABRO1 HTC75 shRNA cell lines were cotransfected with FlagTNKS2 and HA‐K63‐Ub, immunoprecipitated with anti‐Flag, and immunoblotted with anti‐Flag or HA antibodies.

Source data are available online for this figure.