Supplementary Figure 3: Control in vitro experiments.

(a) Strand invasion assays with (UUAGGG)₅ TERRA-like oligonucleotides (10 nM), p-Tel plasmid (0.6 nM) and increasing amounts of the indicated recombinant proteins. Invaded plasmids (inv) were quantified and graphed as the fold increase compared with samples lacking proteins. Values are means ± SD (n = 3 independent experiments). A curve for GST-TRF2 was included as a reference. ss, single stranded oligonucleotides; *, wells. (b) EMSAs with a radiolabeled dsDNA fragment comprising a ~0.8 kb long (TTAGGG)n array (0.03 nM) and increasing amounts of GST-TRF1 or GST-TRF1ΔA, in presence or absence of GST-TRF2. Note that the mobility of GST-TRF2-bound probes is further retarded when GST-TRF1 or GST-TRF1ΔA are included in the reactions. The table on the right is to facilitate direct comparison of DNA and protein concentrations used in EMSAs and strand invasion assays. (c) Strand invasion assays were performed with 0.6 nM p-Tel plasmid, cold (UUAGGG)₅ TERRA-like oligonucleotides (10 nM), and increasing amounts of GST-TRF1 or GST-TRF1ΔA, in presence or absence of GST-TRF2. Reaction products were fractionated in agarose gels, transferred to nitrocellulose membranes and subjected to western blot analysis with anti-TRF1 and anti-TRF2 antibodies. TRF2 signals were quantified and graphed as the fold increase compared to reactions without TRF1 proteins. Values are means ± SD (n = 5 independent experiments). (d) EMSAs with a radiolabeled RNA:DNA duplex comprising 5 TERRA-like RNA repeats and 5 complementary C-rich telomeric DNA repeats. A GST-fusion with the hybrid-binding domain of human RNaseH1 (GST-RH1HBD) was used as a positive control. (e) EMSAs performed with the indicated recombinant proteins (40 nM) and radiolabeled RNA or DNA oligonucleotides (0.25 nM). Bound and free oligonucleotide positions are indicated. *, wells.