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. 2010 Oct 20;107(45):19314–19319. doi: 10.1073/pnas.1010253107

Fig. 2.

Fig. 2.

CBP nuclear exportation on PRL stimulation. (A) In T47D cells treated with PRL for different times (as indicated), CBP was recovered from PRLR immunoprecipitate (IP) for Western blot analysis. (B) Cytosolic (Cyt) and nuclear (Nuc) fractions were prepared from T47D cells treated with PRL for different times, as indicated, and analyzed for the localization of CBP, histone H1, and IκBα. Dynamic CBP subcellular localization was also analyzed with the cytosolic and nuclear fractions prepared from MCF7 cells receiving PRL treatment. (C) CBP was immunostained with monoclonal anti-CBP in T47D cells treated with or without PRL for 60 min. (D) YFP-tagged CBP of WT, NLS1 deletion (NLS1Δ), NSL2 deletion (NLS2Δ), or nuclear export signal deletion (NESΔ) form was transiently expressed in 293T cells and visualized with a confocal microscope. (E) Empty vector (EV) and Myc-tagged PRLR cytoplasmic loop truncation mutants were cotransfected with HA-CBP and coimmunoprecipitated with HA polyclonal antibody. (F) Mass spectrum of the peptide-bearing phospho-T539 of PRLR. (G) Immunoprecipitated PRLR from T47D cells treated with PRL for different times, as indicated, was analyzed by Western blotting with anti-pT539-PRLR. (H) Purified GST-CBP protein was incubated with whole-cell extracts prepared from 293T cells transfected with empty vector (EV) or PRLR-HA (WT, T539A, and T539D). PRLR proteins were recovered from GST-CBP precipitates in Western blots. (I) Indicated GST-CBP domain proteins were purified from bacteria and incubated with Myc-tagged PRLR cytoplasmic domain prepared from 293T transfectants. The CBP-precipitated PRLR cytoplasmic domain was detected by blotting with anti-Myc.