Fig. 5
Plk1-mediated G6PD phosphorylation promotes its dimerization. a, b HeLa cells overexpressing Plk1 (a) or expressing tet-inducible shPlk1 (b) were treated with or without 1 mM disuccinimidyl suberate (DSS), followed by western blot analysis with antibodies against G6PD or Plk1. To induce Plk1 shRNA expression, cells were grown in the presence of 0.1 μg/ml doxycycline for 72 h. β-actin served as loading control. c HeLa cells transfected with eGFP-G6PD and Flag-G6PD vectors were co-transfected with pSin-Plk1 plasmid. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by western blot analysis with antibodies against G6PD or Plk1. d GFP-G6PD and Flag-G6PD co-transfected HeLa cells were treated with BI2536 for 16 h at indicated concentrations. Cells were harvested and subjected to Co-IP using Flag antibody, followed by western blotting with Flag or GFP antibody. e HeLa cells were synchronized using HU double block during which vehicle or 1 μM BI2536 was added 1 h before releasing. Cells were harvested and crosslinked using DSS, followed by western blotting with anti-G6PD. f HeLa cells transfected with eGFP-G6PD and Flag-G6PD were treated with nocodazole. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by western blot analysis with antibodies against G6PD. g NTC- or shPlk1-expressing 293T cells were co-transfected with GFP-G6PD and Flag-G6PD wild type or mutants as indicated. Cells were harvested and subjected to Co-IP using Flag antibody, followed by western blotting with Flag or GFP antibody. h G6PD wild type or its T406A mutant was forced overexpressed in HeLa cells stably expressing shG6PD. Cells were treated with or without 1 mM DSS, followed by western blot analysis with antibodies against G6PD. β-actin served as loading control. NTC denotes non-targeting control
