Journal: Cell Research
Article Title: Ligand stimulation of CD95 induces activation of Plk3 followed by phosphorylation of caspase-8
doi: 10.1038/cr.2016.78
Figure Lengend Snippet: Plk3 interacts with FADD and DISC formation is required for Plk3 activation. (A) HeLa cells were treated with CD95L and CHX for the indicated time periods. Endogenous FADD was immunoprecipitated with rabbit anti-FADD and immunoblotted against Plk3 and FADD. (B) Jurkat cells were treated with CD95L and CHX for the indicated time periods. Cytoplasmic and membrane fractions were separated using hypotonic buffer. Endogenous FADD was immunoprecipitated with rabbit anti-FADD and immunoblotted for Plk3 and FADD. (C) The potential interaction between Plk3 and FADD was monitored via in situ PLA. HeLa cells were treated with CD95L and CHX for the indicated time periods and labeled with the anti-Plk3 (rabbit) and anti-FADD (mouse) antibodies. Single antibody staining (Plk3 or FADD) was used as a control. Scale bar: 10 μm. The quantification is shown on the right. 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 ( * P ≤ 0.05). (D) HeLa cells were transfected with FADD siRNA (siFADD) or a control siRNA (siC) for 48 h followed by the treatment with CD95L and CHX for the indicated time periods. The lysates were immunoblotted against Plk3, FADD, caspase-8, cleaved caspase-8, pT273 caspase-8 and GAPDH. (E) HeLa cells were transfected with FADD siRNA (siFADD) or a control siRNA (siC). Cells were treated with CD95L and CHX for the indicated time periods. The lysates were immunoblotted against FADD (left panel). Immunoprecipitated endogenous Plk3 was incubated with recombinant GST-fused procaspase-8 for an in vitro kinase assay. Samples were immunoblotted against pT273 caspase-8, GST and Plk3 (right panel). (F) HeLa cells were transfected with CD95 siRNA (siCD95) or a control siRNA (siC) and then treated with CD95L and CHX for the indicated time periods. Lysates were immunoblotted against Plk3 and CD95 (upper panel). Immunoprecipitated Plk3 was incubated with GST-fused procaspase-8 for an in vitro kinase assay. Samples were immunoblotted against pT273 caspase-8, GST and Plk3 (lower panel). (G) HeLa Casp8-WT and Casp8-KO cells were treated with CD95L and CHX for the indicated time periods. Lysates were immunoblotted against Plk3, caspase-8, cleaved caspase-8, pT273 caspase-8 and vinculin (upper panel). Immunoprecipitated Plk3 was incubated with GST-fused procaspase-8 for an in vitro kinase assay. Samples were immunoblotted against pT273 caspase-8, GST and Plk3 (lower panel).
Article Snippet: The following antibodies were used according to the respective manufacturers' recommendations — (1) Abcam: Caspase 8-p10 (catalog number ab2553/clone number C502S), pan-Cadherin (ab16505), GAPDH (ab9485), Nucleolin (ab22758), Plk3/PRK (ab33119); (2) BD Biosciences: FADD (556402/A66-2), Fas/ CD95 (610198/13/Fas); (3) BD Pharmingen: Calnexin (610524/37/Calnexin), Plk3 (556518/B37-2); (4) Cell Signaling Technology: Aurora B (3094S), phospho-Chk2 (pT68, 2661), cleaved Caspase-8 (D391/ 18C8), Cyclin B1 (4138S), Fas (8023/4C3), Phospho-p53 (pS20, 9496); (5) Enzo Life Sciences: Caspase-8 (ALX-804-429-C050/ C15); (6) MBL: Lamin B1 (JM-3046-100/ ZL-5); (7) Merck Millipore: Histone H3 (05-499/6.6.2), Plk1 (05-844/35-206), Plk3 (ABS89), Vimentin (MAB3400/V9); (8) R&D Systems: Caspase-8 (AF1650); (9) Santa Cruz Biotechnology: Caspase-8 p18 (sc-6136/C-20), Cdc2 p34 (Cdk1) (sc-54/17), FADD (sc-5559/H-181), GFP (sc-9996/B-2), GST (sc138/B-15); (10) Sigma-Aldrich: β-Actin (A5441/AC-15), Flag tag (FlagM2, F1804), Vinculin (V4505/VIN-11-5).
Techniques: Activation Assay, Immunoprecipitation, In Situ, Labeling, Staining, Transfection, Incubation, Recombinant, In Vitro, Kinase Assay
Santa Cruz Biotechnology is a verified supplier 
![[ A ] Cells were treated with 40 µg/ml concentration of GTP for 1, 5, 10, 15, 30 and 60 min and Western blotting was performed for SAPK/JNK, p-JNK, FAS, <t>FADD,</t> <t>p-FADD,</t> <t>BID</t> and t-BID proteins. A typical actin blot demonstrates internal loading control. [ B ] Cells were treated with 40 µg/ml concentration of GTP for 3, 6, 9, 12, 24 and 48 h and Western blotting was performed for cleaved PARP, procaspase-8, cleaved caspase-8, c-IAP and XIAP proteins. A typical actin blot demonstrates internal loading control. [ C ] Cells were treated with 20 µM concentration of JNK inhibitor SP600125 for 8 h and with 40 µg/ml GTP for 16 h alone, or SP600125 for 8 h followed by GTP treatment in combination followed by Western blotting for JNK, p-JNK, FAS and p-FADD proteins. The expression of native JNK protein was considered as loading control. [ D ] Cell death measurement was performed by photometric enzyme immunoassay Cell Death Detection ELISA kit. The bars represent mean±SD of at least two independent experiments each performed in duplicate, ** p <0.001 represents significant differences as compared to control group. [ E ] Light microscopic images of LNCaPshV and LNCaPshp53 cells treated with SP600125 and GTP alone or in combination. The details are described in the materials and methods section.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7608/pmc03527608/pmc03527608__pone.0052572.g004.jpg)
