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. Author manuscript; available in PMC: 2018 Feb 7.
Published in final edited form as: Nature. 2013 Dec 11;504(7480):401–405. doi: 10.1038/nature12870

Figure 3. Interactions among D3, D14 and D53.

Figure 3

a, In vitro pull-down assay using maltose-binding protein (MBP)–D53, GST–D3 and Strep–D14–His recombinant proteins in the presence or absence of 10 μM GR24. b, Pull-down assays using recombinant GST–D53 or GST–d53 and lysates prepared from Act:D14-GFP/d14 calli in the absence or presence of 20 μM GR24. c, Pull-down assay using recombinant GST–D53 and lysates prepared fromAct:D14-GFP/d14 calli in the presence of GR24 at indicated concentrations. d, Pull-down assay using recombinant GST–D53 and lysates of Act:D14-GFP/d14 calli treated with 20 μM GR24 or SL mimics. e, D53 levels in the 10 μM-GR24-treated calli transformed with D14 mutated at the Ser-His-Asp catalytic triad sites, revealed by immunoblotting with anti-D53 antibodies. D14, Act:D14-GFP/d14; S147A, Act:D14(S147A)-GFP/d14; D268N, Act:D14(D268N)-GFP/d14; H297Y, Act:D14(H297Y)-GFP/d14. f, Pull-down assay using recombinant GST–D53 and lysates of the calli transformed with D14 mutated at its catalytic triad sites in the absence or presence of 20 μM GR24. g, Pull-down assay using recombinant GST–D3 and lysates of Act:D14-GFP/d14 calli in the absence or presence of 20 μM GR24. h, Pull-down assay using recombinant GST–D3 and lysates of transgenic calli transformed with D14 mutated at its catalytic triad sites in the absence or presence of 20 μM GR24. i, Pull-down assay using recombinant GST–D3 and His–Trx–D53 in the absence or presence of 5 μM GR24 or His–Sumo–D14 recombinant proteins. The D14 recombinant proteins in a were detected with anti-His antibody, GFP-fusion proteins in (b–d and f–h) were revealed with anti-GFP antibody, and His–Trx–D53 was detected with anti-D53 antibodies.