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. 2010 Dec 6;286(6):4471–4484. doi: 10.1074/jbc.M110.181511

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 1. — In vivo expression and analysis of FLAG-gTTLL proteins in G. duodenalis. A, expression profile of the FLAG-tagged recombinant proteins and their effect on the g14-3-3 is shown. 20 μg of trophozoite protein lysate were separated on 4–12% SDS-PAGE and blotted on a nitrocellulose membrane. The membrane was incubated with the indicated antibodies (bottom). Expected molecular size of the indicated TTLLs is reported on the right of the upper panel. Molecular size markers are on the left. Different time exposition of the same filter incubated with α-FLAG Ab are presented and separated by lines. The equal loading of the sample material is demonstrated by immunostaining with the anti-α-tubulin (panel α-αTUB). B, affinity purification of g14-3-3 is shown. Soluble proteins from trophozoites were incubated with GST-difopein and eluted with 2 mm A8Ap phosphopeptide. An aliquot (1/20) was analyzed in duplicate on 4–12% gradient NuPAGE. Upper panel, a Coomassie-stained gel is shown. Lower panel, immunoblotting with rabbit anti-g14-3-3 serum is shown. Molecular size markers are on the left. The position of the upper and the lower bands, used for MALDI analyses, is indicated. C, mass spectrometry analysis of g14-3-3 is shown. A zoom view of MALDI spectra of g14-3-3 from WBC6 wild type strain and FLAG-gTTL3 transfectant after tryptic digestion of the lower bands (spectra on the left) and of the upper bands (spectra on the right) is shown. m/z ranges have been selected to compare the distribution and the length of polyGly chains. The number of the glycines added to the 230–248 peptide and deduced from the m/z value is reported. The position of the peptide 230–248 is indicated in bold in the g14-3-3 protein sequence (bottom). D, immunopurification of the FLAG-tagged proteins is shown. An aliquot (1/20) of the FLAG peptide-eluted material (WBC6 or FLAG-gTTLL3 transfectant) was separated by 4–12% SDS-PAGE, transferred to a nitrocellulose membrane, and immunostained with α-FLAG mAb. The position of the FLAG-tagged protein is indicated on the right. E, an in vitro glycylation assay is shown. 20 μl of affinity-purified FLAG-gTTLL3 or control immunopurification from WBC6 strain were incubated where indicated with 0.25 μg of recombinant Prescission-cleaved g14-3-3 (Rec. g14-3-3) and separated on 12% SDS-PAGE, transferred on nitrocellulose, and subjected to Western blot with mouse AXO49 Ab (left panel) and then with rabbit α-g14-3-3 serum (right panel). The arrow indicates the molecular size of recombinant g14-3-3. The asterisk on the left indicates the small subunit of the mouse anti-FLAG mAb cross-reacting with the secondary anti-mouse Ab used to detect AXO49.