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. 2011 Aug 5;19(2):321–332. doi: 10.1038/cdd.2011.101

Figure 1.

Figure 1

Plk1 interacts with TNKS1 in vitro and in vivo. (a) Lysates from control AGS (a gastric cancer cell line) cells and ones stably overexpressing HA-tagged Plk1 (AGS-HA-Plk1) were prepared for immunoprecipitation using an anti-HA antibody and subjected to immunoblot analysis. The arrowhead and the asterisk indicate HA-PlK1 and IgG heavy chains, respectively. (b) Lysates from the AGS-HA-Plk1 cells were used for immunoprecipitaion using an anti-HA antibody. The eluted HA-Plk1 immune complexes were analyzed by SDS-PAGE and subsequent Coomassie staining followed by mass spectrometric analysis. TNKS1, HA-Plk1, and TCTP (as a positive control) polypeptides are indicated by arrows. (c) Schematic diagrams of TNKS1. HPS, homopolymeric tracts of histidine, proline, and serine repeats; ANK, ankyrin domain; SAM, sterile alpha motif; PARP, poly(ADP-ribose) polymerase. Purified His-tagged Plk1 were incubated with bead-bound GST or GST-TNKS1 (GST-TNKS1) fusion proteins. The eluted GST and GST fusion proteins were resolved by SDS-PAGE prior to immunoblot analyses. (d) Schematic diagrams of Plk1. GST and GST-Plk1 fusion proteins were incubated with HeLa cell lysates cultured in the presence of nocodazole for 12 h. Bead-bound GST or GST-Plk1s were washed, resolved by SDS-PAGE, and immunoblotted using anti-TNKS1 antibody. (e) HeLa cells were cultured in the absence (Asyn) or presence (Noco) of nocodazole. Lysates were incubated with purified GST or GST-Plk1 fusion protein. Bead-bound GST or GST-Plk1s were washed, resolved by SDS-PAGE, and immunoblotted using anti-TNKS1 antibody. (f) HeLa cells were cultured in the absence (Asyn) or presence (Noco) of nocodazole. Lysates were immunoprecipitaed using normal IgG (negative control) or anti-Plk1 antibody. The immunocomplexes were resolved by SDS-PAGE and immunoblotted using and-Plk1, anti-TNKS1, and anti-actin antibodies. (g) Extracts were prepared from HeLa cells synchronized at mitosis by nocodazole, and fractionated by gel filtration using a Superdex-200 column. Fractions (numbered 7–24) were analyzed by immunoblotting using antibodies against TNKS1 (TNKS1), NuMA, PARP, Plk1, and Aurora-A (Aur-A). Input indicates 4% of the extract