Figure 2.

Plk3 and procaspase-8 interact. (A) HEK 293T cells were co-transfected with GFP-Plk3 or GFP-Rip3 and Flag-procaspase-8. GFP-Rip3 was used as a positive control for the co-IP with procaspase-8. Precipitates were immunoblotted for Flag and GFP. (B) The binding ability of GST-fused procaspase-8 was analyzed in pull-down assays. GST-procaspase-8 or GST alone (as a control) was incubated with HeLa cell lysates. The pull-down was analyzed by immunoblotting for Plk3 and GST. (C) Schematic representation of recombinant GST-fused full-length Plk3 (GST-Plk3-WT) and truncated forms of Plk3 (GST-Plk3-NT1, -NT3 and -CT1) used in pull-down assays. (D) GST-Plk3-WT or Plk3 subdomains were incubated with HeLa cell lysates for pull-down assays. The immunoblot analysis was performed using caspase-8- and GST-antibodies. (E) HeLa cells were treated with 50 ng/ml nocodazole (Noc) for 16 h to enrich for mitotic cells. Endogenous caspase-8 was immunoprecipitated from cell lysates. Normal mouse IgG was used as a control. The immunoprecipitates and the lysates were immunoblotted for caspase-8, Plk1, Cdk1, Cyclin B1, Plk3 and Aurora B. (F) HeLa cells were treated with 50 ng/mL CD95L and CHX for 0 h and 2 h. Endogenous caspase-8 was immunoprecipitated from cell lysates with anti-caspase-8 p18 goat antibody. Normal goat IgG (Santa Cruz) was used as a control. The immunoprecipitates and the lysates were immunoblotted for Plk3, caspase-8, cleaved caspase-8 and p10. (G) The interaction of Plk3 and procaspase-8 (Casp8) was monitored via in situ PLA. HeLa cells were labeled with the anti-Plk3 (Abcam, rabbit) and anti-Casp8 (mouse) (left panel) or anti-Plk3 (BD, mouse) and anti-Casp8 (rabbit) (right panel) antibodies. Single antibody staining (Plk3 or Casp8) was used as a control. Scale bar: 10 μm. Each bar represents the mean value ± SD (n = 3). The differences between single and double antibody staining were statistically significant by Student's t-test (lower panels) (^*P ≤ 0.05; ^**P ≤ 0.005).