Fig. 2: TRF1 is PARylated by PARP1 in-vitro and in-vivo.

a Covalent PARylation assay was performed by incubating a high activity purified PARP1 enzyme in the NAD+ containing PARylation buffer in absence or presence of recombinant His-tag full length TRF1, activated DNA was added to further stimulate the PARylation reaction. b The PARylation reaction was performed as above described in presence of biotynilated NAD+. In both a and b Protein mixtures were resolved on SDS-PAGE and incubated with an anti-PAR (a) or anti-Streptavidin (b) antibodies, to detect polymers, or Anti-TRF1. Signals were revealed by chemiluminescence. c Noncovalent PARylation assay: increasing quantities of recombinant full length TRF1 or H1 histone were spotted on nitrocellulose by dot blot, incubated with previously synthetized and purified PARs, and then incubated with anti-PAR antibody (to detect bound polymers). Pictures are representative of three independent experiments with similar results. d, e HeLa cells were transfected as indicated, then synchronized as previously described, collected and immunoprecipitated with the indicated antibodies or IgG as control. Input and immunoprecipitated samples were processed for Western blot analysis of the indicated antigens. Representative images of three independent experiments are shown. Western blot signals were quantified by densitometry, and the mean of three independent experiments is shown in the histograms, bars are SD.