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. 2016 Sep 13;5:e17523. doi: 10.7554/eLife.17523

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© 2016, Pylypenko et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 2. — (A) Co-immunoprecipitation assay of HEK293 cells transfected with plasmids expressing AcGFP (GFP), AcGFP-tagged MyoVb deletion mutants and indicated Myc-epitope tagged Spir-1 and Spir-2 proteins. Cell lysates were immunoprecipitated with anti-Myc antibodies. The cell lysates (input) and immunoprecipitates (co-IP) were analyzed by immunoblotting with antibodies as indicated. The Spir-2-GTBM and the MyoVb globular tail domain (GTD) were identified as being essential for the Spir/MyoV interaction. N = 4 experimental repeats. WB, Western blotting. (B) GST-pulldown studies employing purified bacterially expressed recombinant proteins to analyze a direct interaction of Spir-2-linker and MyoVa/Vb-GTD. The mutation of two highly conserved leucines within the Spir-2-GTBM to alanines (human Spir-2-L408A, L409A; Spir-2-linker-LALA, see also Figure 4A) largely impairs binding of this mutant to GST-MyoVa/Vb-GTD. N = 4 experimental repeats. (C) Fluorescence spectroscopy was used to determine the dissociation constants (K_d) for MyoVa and MyoVb GTD binding to His₆-mCherry-Spir-2-linker. Error bars represent SEM (n = 4 experimental repeats).

DOI: http://dx.doi.org/10.7554/eLife.17523.003

Figure 2—figure supplement 1. — (A) Co-immunoprecipitation assay of HEK293 cells transfected with plasmids expressing AcGFP (GFP), AcGFP-tagged MyoVb deletion mutants and indicated Myc-epitope tagged Spir-1 and Spir-2 proteins. Cell lysates were immunoprecipitated with anti-Myc antibodies. The cell lysates (input) and immunoprecipitates (co-IP) were analyzed by immunoblotting with antibodies as indicated. The Spir-2-GTBM and the MyoVb globular tail domain (GTD) were identified as being essential for the Spir/MyoV interaction. N = 4 experimental repeats. WB, Western blotting. (B) GST-pulldown studies employing purified bacterially expressed recombinant proteins to analyze a direct interaction of Spir-2-linker and MyoVa/Vb-GTD. The mutation of two highly conserved leucines within the Spir-2-GTBM to alanines (human Spir-2-L408A, L409A; Spir-2-linker-LALA, see also Figure 4A) largely impairs binding of this mutant to GST-MyoVa/Vb-GTD. N = 4 experimental repeats. (C) Fluorescence spectroscopy was used to determine the dissociation constants (K_d) for MyoVa and MyoVb GTD binding to His₆-mCherry-Spir-2-linker. Error bars represent SEM (n = 4 experimental repeats).

DOI: http://dx.doi.org/10.7554/eLife.17523.003