A Functional Nuclear EGFR, Src and Stat3 Complex Figure 4. Studies of protein complex and protein binding partners using the Detection and Analysis through Nanoparticle Sizing technology. (A) Kinetic binding assay of EGFR-gold nanoparticle (GNP) probe (or mouse IgG1-GNP probe as negative control) binding to (i) EGFR protein and its complex from Panc-1 nuclear extracts, and the (ii) inhibitory effect of the mouse monoclonal anti-EGFR antibody on the EGFR-GNP probe binding to the EGFR protein; and (B) Protein complex binding partner analysis whereby the polyclonal anti-Stat3, anti-Src or anti-EGFR antibody or the non-specific rabbit IgG (negative control) is added to the assay solution prepared from the (i) non-specific mouse IgG1-GNP probe (negative control), or (ii) anti-EGFR-GNP probe; Data are representative of 4 independent studies. doi:10.1371/journal.pone.0019605.g004 EGFR-GNP probe in the detection of EGFR from nuclear extract, an inhibition experiment was conducted in which the extract was first treated with the monoclonal anti-EGFR antibody. It is expected that the binding between the monoclonal antibody and the EGFR protein from the sample will block the subsequent binding of EGFR (in the sample) to the GNP immunoprobes, therefore, leading to decreased nanoparticle size increase during the kinetic assay. Data shown in Figure 4A(ii) confirmed the inhibitory effect of the monoclonal anti-EGFR antibody. The magnitude of this inhibition was correlated to the sample treatment time: the 24- min-treatment inhibited the particle size increase in the assay stronger than the 7 min-treatment (Fig. 4A(ii)). After using the anti-EGFR-GNP probe to ‘‘catch’’ the EGFR protein or protein complex from the nuclear extract sample, a second polyclonal antibody (anti-Stat3, anti-Src, or anti-EGFR antibody, or non-specific rabbit polyclonal IgG) was then added to the assay solution to identify the binding partner of the complex. In negative control studies using the mouse IgG1-GNP probe, particle size remains nearly unchanged after the addition of the four polyclonal antibodies (Fig. 4B(i)). This result confirmed the kinetic binding study that the non-specific mouse IgG1-GNP probe did not bind with the EGFR protein or protein complex from the sample. The addition of a polyclonal antibody itself to the assay solution did not cause significant nanoparticle size change. By contrast, with the anti-EGFR-GNP probe, significant particle size increase was observed when anti-Stat3 or anti-Src antibody was added to the assay solution (Fig. 4B(ii), anti- Stat3, Src). A smaller size increase was observed from anti-EGFR antibody (Fig. 4B(ii), anti-EGFR) and the rabbit IgG (Fig. 4B(ii), IgG). Per the assay principle (see Fig. S2), the substantial particle size increases observed from the addition of anti-Stat3 or anti-Src antibody to the assay solution can only be explained by the presence of EGFR in complex with Stat3 and Src in the nuclear extract. EGFR was specifically bound to the nanoparticle immunoprobes, bringing along the Stat3 and Src proteins to the nanoparticle surface, and the subsequent incubation of this assay solution with anti-Stat3 or anti-Src antibody led to further increase of the nanoparticle size. Furthermore, it appears that there is an equal amount of Stat3 and Src proteins in the EGFR, Src, and Stat3 complex. In multiple assays conducted so far, the size increase caused by anti-Src antibody is always just slightly lower than the case of anti-Stat3 antibody. Src is a smaller protein (60 KDa) than Stat3 (89–90 KDa). Therefore, with the binding of the same amount of antibody to the protein complex-GNP conjugates, the particle size should be just slightly smaller in the case of anti-Src assay solution than anti-Stat3 assay solution. Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG. This is explained by the fact that Stat3 and Src are located on the surface of the nanoparticle-bound EGFR protein, therefore, block the binding of anti-EGFR antibody to the bound EGFR proteins. The effectiveness of polyclonal anti-EGFR antibody with the EGFR protein in the sample was confirmed in a different PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e19605