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. 2011 Nov 28;40(6):2445–2453. doi: 10.1093/nar/gkr1165

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© The Author(s) 2011. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — STAT is associated with SAYP-containing complex. (A) Western blot analysis of total proteins from embryos or S2 cells with antibodies against STAT. To confirm their specificity, lysate of S2 cells expressing HA-tagged STAT was stained with anti-HA antibodies. Staining for tubulin and TBP is shown as a loading control. (B) Fractions after gel filtration of DNase I-treated nuclear extracts from embryos on Superose 6 were analyzed for the presence of SAYP and STAT proteins. STAT was detected not only as a free protein (fractions 32–34) but also in association with high-MW complexes, together with SAYP (fractions 16–17). (C) STAT and SAYP co-immunoprecipitated with each other from gel filtration fractions 16–17 of the extracts from embryos. IP with pre-immune IgG was used as a negative control. Equal portions of the input (In) and precipitated (IP) material were tested. (D) Antibodies against STAT co-immunoprecipitated MOR (component of Brahma) and TAF1 (component of TFIID) from gel filtration fractions 16–17. (E) Recombinant Flag-SAYP and HA-STAT co-immunoprecipitated with each other. Both proteins were co-expressed in cells, then IP with antibodies against either FLAG or HA was performed. In control IP, only one recombinant protein was expressed. Equal portions of the input (In) and precipitated (IP) material were tested. (F) Scheme of SAYP and STAT proteins. Boundaries of the tested regions and the total protein length (number of amino acids) are indicated. (G) Recombinant Flag-Gal4-fusions of SAYP fragments and the HA-tagged activation domain of STAT were tested for co-immunoprecipitation (the left column). Both proteins were co-expressed in cells, then IP with antibodies against HA was performed. In control IP, only Flag-tagged recombinant protein was expressed. Recombinant Flag-SP fragment co-immunoprecipitated HA-AD of STAT and HA-STAT (the right column). Equal amounts of the input (In) and precipitated (IP) material were tested.