Figure 3

FANCD2 binding to TNKS1. (A) HeLa cell extracts were immunoprecipitated either with anti-tankyrase 1 (TNKS1) or anti-tankyrase 2 (TNKS2) antibody followed by Western blotting and probing with anti-FANCD2 antibodies. In parallel, same amount of cell lysate was immunoproecipitated with anti-FANCD2 antibodies and corresponding Western blot was probed with TNKS1 antibodies. Heavy and light IgG chains are depicted by arrows to show equal loading. (B) In-vitro pull-down assay showing binding of recombinant FANCD2 protein to TNKS1 was performed with 5 ug of His-tagged TNKS1 (INP, input of half TNKS1 amount used for binding) immobilized onto resin followed by incubation either with recombinant (B) or with heat-inactivated FANCD2 (C). The beads were washed with TNE buffer (fractions W1-3) followed by several elution steps (E1-5). Corresponding aliquots from each step (1/4 volume each) were subjected to Western blot analyses followed by probing with anti-FANCD2 antibodies. Densitometric measurement of the Western blot signals was performed by TotaLab2.0 software and corresponding intensities from the all fractions versus input were calculated as percentages of retained proteins.