A Functional Nuclear EGFR, Src and Stat3 Complex Figure 3. Co-immunoprecipitation with immunoblotting analysis of the effects of modulation of EGFR, Src and Stat3 on the nuclear EGFR, Src and Stat3 complex. (A, B, and C) Immunoblotting analyses of immunecomplexes of Stat3 (IP:Stat3), EGFR (IP:EGFR), or Src (IP:Src) prepared from nuclear extracts of Panc-1 cells untransfected or transfected with Src siRNA, EGFR siRNA, or control (con) siRNA (A), or treated with or without the EGFR inhibitor (ZD1839, ZD), Src inhibitor (Dasatinib, Das), or the Stat3 inhibitor (S3I-201) for 1 or 24 h (B), or from nuclear extracts pre- incubated for 2 h with or without 100 mM pY1068, pY1086, or SPI peptide (C) and probing for EGFR, Src, Stat3; or (D) immunoblotting analysis of nuclear extracts prepared from Panc-1 cells treated or untreated with phenylarsine oxide (PAO) and probing for Src, Stat3, EGFR. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate or nuclear extract used in the assay; Data are representative of 3 independent studies. doi:10.1371/journal.pone.0019605.g003 Nuclear EGFR, Src and Stat3 heterocomplex is independent of EGFR-mediated endocytosis We were interested to determine how the known nuclear translocation mechanisms might affect the nuclear presence of the heteromeric EGFR, Src and Stat3 complex. Several mechanisms have been reported for EGFR nuclear translocation, including receptor endocytosis, endosomal sorting machinery, importins alpha1/beta1, and exportin CRM1 [30,31]. Previous studies have also shown that Stat3 nuclear translocation is facilitated by EGFR-mediated endocytosis [6]. However, the siRNA knockdown of EGFR did not affect the nuclear presence of Src/Stat3 (Fig. 3A(ii)), suggesting EGFR-mediated mechanisms may not be utilized for the nuclear localization of Stat3 or Src. To further determine whether endocytosis is required for the nuclear presence of the heteromeric complex, cells were treated with the pharmacological inhibitor of endocytosis, phenylarsine oxide [6]. In contrast to PAO-induced inhibition of nuclear Stat3 DNA-binding activity induced by EGF [6], immunoblotting analysis showed treatment with PAO has no effect on the nuclear presence of EGFR, Src and Stat3 heterocomplex in pancreatic cancer cells (Fig. 3D), suggesting the nuclear presence of the heteromeric complex is not dependent on endocytosis. Altogeth- er, these findings show that EGFR, Src and Stat3 associate into a complex in a manner where all three proteins interact. However, our study does not exclude the possibility that other proteins could be present in the complex. Detection and Analysis of the EGFR, Src and Stat3 heterocomplex through Nanoparticle Sizing (DANS) technology with Dynamic Light Scattering The EGFR, Src and Stat3 association was further probed using DANS (Detection and Analysis through Nanoparticle Sizing) technology [32,33,34,35,36,37]. The principle of DANS technology for protein complex detection and binding partner analysis is a single-step solution immunoassay based on gold nanoparticle (GNP) immunoprobes coupled with dynamic light scattering (DLS) detection. Details of this technology are provided in ‘‘Text S1’’ and illustrated in Fig. S2. Based on this general principle, the assay is designed to detect and identify protein binding partners of a protein complex. The kinetic binding assay revealed and confirmed that the anti-EGFR-GNP probe detects EGFR from the sample specifically (Fig. 4A(i)). The increase in particle size obtained with the anti- EGFR-GNP probe was much larger than the one obtained with the non-specific mouse IgG1 control probe (Fig. 4A(i)). The approxi- mate net increase of the average nanoparticle size of the assay solution was about 70 nm, after deducting the size increase caused by non-specific interactions. For the mouse IgG1-GNP probe, the particle size increase was less than 10 nm after incubation with the sample for 30 min. Furthermore, the steepness of the kinetic curve for the EGFR probe suggested specific interactions in the assay system, while the shallow curve for the mouse IgG1 probe indicated the small size increase of the nanoparticle probes was due to non- specific interactions. To further confirm the specificity of anti- PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19605