Fig 4.

Association of GMD with tankyrase 1 is lost at mitosis. (A and B) Immunoprecipitation analysis across the cell cycle. HeLaI.2.11 cells were synchronized in G₁/S by a double thymidine block, released, and collected at 2-h intervals, and whole-cell extracts (Input) generated in TNE buffer were analyzed by immunoblotting with antibodies to TNKS 762, GMD, NuMA, and α-tubulin. *, more slowly migrating form of tankyrase 1 that marks entry into mitosis at the 10- and 12-h time points. (B) Staged extracts generated in TNE buffer were immunoprecipitated with anti-TNKS1 762 and analyzed by immunoblotting with antibodies to TNKS 762, GMD, and NuMA. (C) Immunoprecipitation analysis in cells arrested in S phase versus mitosis. HelaI.2.11 cells were untreated (−) or incubated with hydroxy urea (HU) or nocodazole (Noc) for 20 h. Cell extracts generated in buffer C were analyzed directly (Input) or were immunoprecipitated with control (C) or anti-TNKS1 762 antibody and analyzed by immunoblotting with antibodies to TNKS1 762, GMD, or NuMA. A shorter exposure of the GMD blot is indicated. (D) HeLaI.2.11 cells were synchronized in G₁/S by a double thymidine block, released into nocodazole, and collected at 2-h intervals. Staged extracts generated in buffer C were immunoprecipitated with anti-TNKS1 762 and analyzed by immunoblotting with antibodies to TNKS 762 and GMD. GMD levels relative to tankyrase 1 and normalized to the zero time point are indicated below the blot.