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. 2015 Aug 15;26(16):2939–2954. doi: 10.1091/mbc.E15-04-0247

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© 2015 Barbosa, Carreira, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 7: — Direct phosphorylation-dependent interaction between CREB-H and Fbw1a. Cells were transfected with Flag-tagged Fbw1a and soluble extracts made. Equal amounts of purified GST, GST-wt, or GST-DSG protein bound to glutathione-agarose beads were incubated without (–) or with (+) purified GSK-3 and CKII for 30 min before the addition of the Fbw1a- transfected soluble extracts. After further incubation, the beads were isolated, washed extensively, and then analyzed by SDS–PAGE and Western blotting for Fbw1a and for GST fusion protein in the pull-down (Fbw1a and GST panels). The input sample (Inp) represents 1/50 of the total input, and 1/5 of the pull-down material was analyzed in each case. Equal amounts of total GST-fusion proteins were incubated in each case and present in the pull-down as detected by anti-GST antibody (bottom GST panel). Specific interaction with wt was observed but only after phosphorylation by GSK-3 and CKII. In parallel, no significant interaction was observed for the GST control or for GST-DSG.