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. 2007 Apr 13;98(6):850–857. doi: 10.1111/j.1349-7006.2007.00462.x

Figure 2.

Figure 2

Tankyrase 1 has no physical interaction with mouse telomeric repeat binding factor 1 (TRF1). (a) In vitro pull‐down assay of tankyrase 1 and TRF1. Human (left upper panel) and mouse (left lower panel) tankyrase 1 were prepared by in vitro translation and incubated with glutathione beads coupled with glutathione S‐transferase (GST)‐fused proteins as indicated. Coomassie Brilliant Blue (CBB)‐stained gel corresponding to the left upper panel is shown in the right panel. The bead‐bound tankyrase 1 was detected with western blot analysis. Molecular mass markers (K) are indicated at the left. (b) Partial amino acid sequences of human and mouse tankyrase‐binding protein of 182 kDa (TAB182). Each protein has a canonical tankyrase 1 binding motif, RPQPDG. Numbers indicate the positions of amino acid residues in the human paralog. (c) In vitro pull‐down assay of tankyrase 1 and TAB182. Human (H) and mouse (M) tankyrase 1 were prepared and incubated with glutathione beads coupled with GST‐fused proteins, as indicated. The bead‐bound tankyrase 1 was detected as in (a). (d) In vitro poly (ADP‐ribose) polymerase (PARP) assay. Tankyrase 1 was affinity‐purified from HTC75/FN‐tankyrase 1 (FLAG‐nuclear localization signal [NLS]‐tankyrase 1) cells( 13 ) and subjected to the PARP assay described in Materials and Methods. GST‐fused mouse and human TRF1 were used as acceptors for tankyrase 1's substrate, [32P]‐nicotinamide adenine dinucleotide (NAD). Reactions were carried out in the absence or the presence of 3 mM 3‐aminobenzamide (3AB), a PARP inhibitor, fractionated on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and visualized by CBB staining (upper panel) or autoradiography (lower panel). Note that tankyrase 1 is also ADP‐ribosylated by itself.( 6 ) IP, immunoprecipitated; IgH, immunoglobulin heavy chain. (e) Co‐immunoprecipitation assay. HeLa I.2.11 cells were transiently transfected with Myc‐mTRF1 or Myc‐hTRF1. The cell lysates were prepared and immunoprecipitated with anti‐Myc antibody (9E10). The immunocomplex was subjected to western blot analysis with anti‐tankyrase antibody (H‐350).