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. 2014 Feb 19;5:3351. doi: 10.1038/ncomms4351

Figure 2. Isolation of DUSP26 as a FADD phosphatase and an AK2-binding partner.

Figure 2

(a) HEK293T cells were co-transfected with p-FADD-HA and each phosphatase cDNA for 24 h, cell extracts were subjected to western blotting using anti-p-FADD and anti-FADD antibodies and to phosphatase assays with pNPP. Bars represent mean±s.d. (n=3). (b) HEK293T cells were co-transfected with FADD-HA and pcDNA3, Flag-DUSP26 or Flag-DUSP26 C152S for 24 h, and cell extracts were then analysed by western blotting. (c) HeLa cells were transfected with pSuper, pDUSP26 or pDUSP26 shRNA for 36 h, after which cell extracts were subjected to western blotting using anti-p-FADD, anti-FADD and anti-α-tubulin antibodies. Total RNAs were purified and analysed with RT–PCR using DUSP26- or GAPDH-specific synthetic oligonucleotides as primers. (d) HEK293T cells were transfected with p3 × Flag or p3 × Flag-DUSP26 for 36 h, and cell extracts were prepared and subjected to the pull-down assay using anti-FLAG agarose-beads. The bound proteins were resolved by SDS–PAGE, stained with Coomassie-blue, and analysed by LC-MS/MS or analysed by western blotting using the indicated antibodies. NS indicates non-specific signal. (e) HEK293T cells were co-transfected with pAK2-HA and pFlag-DUSP26 for 36 h and cell extracts were subjected to immunoprecipitation (IP) assay using anti-HA or anti-Flag antibody. Whole-cell lysates and the immunoprecipitates were probed by western blotting with anti-FLAG or anti-HA antibody. (f) HeLa cell extracts were subjected to IP analysis using anti-AK2 or anti-DUSP26 antibodies and then the immunoprecipitates were analysed by western blotting with the indicated antibodies. (g) AK2 over-expression enhances the binding of DUSP26 to FADD. HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either GFP or AK2-GFP for 36 h, after which cells extracts were subjected to IP analysis using anti-HA antibody. Expression levels of Flag-DUSP26, FADD-HA, GFP and AK2-GFP in whole-cell lysates were examined by western blotting with the indicated antibodies. (h) HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either pSuper or AK2 shRNA for 36 h. Cell lysates were subjected to IP analysis with anti-HA antibody, and expression levels of Flag-DUSP26, FADD-HA and AK2 in whole-cell lysates were examined by western blotting.