Fig. 4.
SA1 associates with telomeres in vivo. (A,B) Telomeric ChIP analysis of TRF1 and SA1. (A) Autoradiograph showing telomeric DNA ChIP analysis of HeLaI.2.11 cells using the indicated antibodies. Dot blots with the immunoprecipitated DNA were analyzed by Southern blotting with 32P-labelled telomeric (Telo) or Alu repeat (Alu) probes. (B) Graphical representation of the percentage of immunoprecipitated Telo or Alu DNA relative to total input DNA (as in A), derived from four independent experiments. Values are means ± s.d. (C–E) Ablation of the SA1 signal at telomeres by SA1 siRNA. HelaI.2.11 cells were transfected with siRNA to GFP or SA1 for 48 hours and analyzed by (C) immunoblotting with antibodies against SA1 or α-tubulin and (D) telomeric ChIP with antibodies against TRF1 or SA1. (E) Graphical representation of the percentage of SA1-immunoprecipitated Telo or Alu DNA relative to total input DNA (as in D). (F–H) SA1 is reduced at telomeres in mitosis. HeLaI.2.11 cells were synchronized by a double thymidine block, released, and collected by trypsinization at 0 hours (G1/S) or 10 hours (G2/M) and analyzed by (F) FACS (y-axis, cell numbers; x-axis, relative DNA content based on propidium iodide staining) and (G) telomeric ChIP with antibodies against TRF1 or SA1. (H) Graphical representation of the percentage of SA1-immunoprecipitated Telo or Alu DNA relative to total input DNA (as in G). Values are means ± s.e.m., derived from two independent experiments. (I–K) SA1 is increased at telomeres in TNKS1-depleted mitotic cells. HelaI.2.11 cells were transfected with siRNA to GFP or TNKS1 for 48 hours, isolated by mitotic shake-off, and analyzed by (I) immunoblotting with antibodies against TNKS1, SA1 or α-tubulin and (J) telomeric ChIP with antibodies against TRF1 or SA1. (K) Graphical representation of the percentage of SA1-immunoprecipitated Telo or Alu DNA relative to total input DNA (as in J). Values are means ± s.e.m., derived from two independent experiments.