Dynamic Light Scattering (DLS) measurement (Hellma cuvette QS 3 mm), 20 ml of the anti-EGFR-GNP probe was mixed with 2 ml of the sample, and the particle size increase was read with a DLS instrument (Zetasizer Nano ZS90 DLS system, Malvern Instruments Ltd, England) at exactly 1, 6, 11, 16, and 30 min after the mixing. The same experiment was also performed using the mouse IgG1-GNP probe. In order to confirm the specificity of the anti-EGFR-GNP probe in the detection of EGFR from nuclear extract, an inhibition experiment was conducted by treating 5 ml of the sample with 1 ml of monoclonal anti-EGFR antibody at room temperature for 7 min and 24 min prior to using the sample in the assay. After this incubation, 20 ml of the anti-EGFR-GNP probe was mixed with 2 ml of the treated sample, and the particle size was read at 1, 6 and 11 min after the mixing. Protein complex binding partner study using polyclonal antibody In a 1.5 ml microcentrifuge tube, 80 ml of the anti-EGFRGNP probe was mixed with 8 ml of the sample. After incubation for 30 min at room temperature, this solution was divided into four 20 ml portions. After transferring into the sample cell, the particle size of each of these portions was read with a DLS instrument. After this reading, the solution was spiked with a polyclonal antibody: either with 1 ml of anti-Stat3 or 2 ml of anti-Src or 1 ml of anti-EGFR or 0.5 ml of rabbit IgG. The particle size increase was read at exactly 5 min and 10 min after the start of the first reading. Dynamic Light Scattering (DLS) measurements The DLS measurements of all sample solutions were conducted using a Zetasizer Nano ZS90 DLS system equipped with a red (633 nm) laser and an Avalanche photodiode detector (APD) (quantum efficiency.50% at 633 nm) (Malvern Instruments Ltd). DTS applications 5.10 software was used to analyze the data. The average particle size (Z-average) of the solution was obtained using a Cumulant method. For each sample, ten DLS measurements were conducted with one run, and each run lasted for 10 seconds. All measurements were done at a 90u detection angle. Results Detection of nuclear EGFR, Src and Stat3 heterocomplex We sought to investigate the signaling complex assembly and the dynamics of the interactions of the hyperactive Stat3, EGFR and Src [14,16,23] within the context of the human pancreatic cancer phenotype. Co-immunoprecipitation (co-IP) with immu- noblotting analysis shows, EGFR immunecomplex from Panc-1 or Colo-357 whole-cell lysates contained both Src and Stat3 (Fig. 1A(i)), Src immunecomplex contained both EGFR and Stat3 (Fig. 1A(ii)), while Stat3 immunoprecipitate contained EGFR and Src (Fig. 1A(iii)). For specificity, the non-specific rabbit IgG in the immunoprecipitation and immunoblotting analysis for Src, Stat3 or EGFR showed no detectable protein (Fig. 1A, IgG, and data not shown), and the immunoblotting analysis performed on the immune precipitates in the presence of the respective blocking peptides (BP) showed a complete or near complete block of the immune detection of Stat3, Src or EGFR, compared to the levels detected in the absence of the blocking peptides (Fig. 1B(i)-(iii), compare + BP, lower panels to - BP, upper panels). We determined the effect of siRNA knockdown of EGFR or Src on the complex formation. Co-immunoprecipitation with immuno- blotting studies of whole-cell lysates from Panc-1 cells showed that when EGFR is knockdown by siRNA (Fig.1C(i), upper band), Src immunecomplex remains associated with Stat3 (Fig. 1C(i), IP:Src), A Functional Nuclear EGFR, Src and Stat3 Complex and vice-versa (Fig. 1C(i), IP:Stat3). Likewise, when Src is knockdown by siRNA (Fig. 1C(ii), upper band), EGFR immune- complex remains associated with Stat3 (Fig. 1C(ii), IP:EGFR), and vice-versa (Fig. 1C(ii), IP:Stat3). Scrambled (con) siRNA has no effect. These findings indicate that with respect to the EGFR, Src and Stat3 heteromeric complex, Stat3 proteins remains associated with Src or EGFR, respectively, upon the siRNA knockdown of EGFR or Src. We asked the question whether the observed EGFR, Src and Stat3 heteromeric complex was present in the nucleus. Co- immunoprecipitation with immunoblotting analysis of nuclear extracts shows that EGFR immunecomplex contained both Stat3 and Src (Fig. 2A(i), IP:EGFR), Src immunecomplex contained both Stat3 and EGFR (Fig. 2A(ii), IP:Src), while Stat3 immune- complex contained both EGFR and Src (Fig. 2A(iii), IP:Stat3). These data demonstrated the presence of the EGFR, Src and Stat3 heteromeric complex in the nucleus. For specificity of the immunoreagents, the non-specific rabbit IgG pull-down samples that were similarly immunoblotted showed no detectable EGFR, Src or Stat3 (Fig. 2A, IgG and data not shown). Immunoblotting analysis probing for the Tata-binding protein (TBP) confirmed that the extracts used in these studies are of nuclear origin (Fig. 2A(iv)). The heteromeric complex was validated by performing sequential immunoprecipitation analysis, whereby EGFR immunecomplex (IP:EGFR) was further subjected to a secondary immunoprecipitation using anti-Src antibody (IP:EGFR/IP:Src) and then immunoblotted for EGFR and Stat3. The results of these studies showed the presence of EGFR and Stat3 in the sequential immunoprecipitates (Fig. 2B, IP:EGFR/ IP:Src). By contrast, IgG pull-down that was subjected to immunoblotting for EGFR or Stat3 showed no detectable levels (Fig. 2B, IgG), further confirming the specificity of the immunor- eagents used. The presence of the EGFR, Src and Stat3 complex in the nucleus of pancreatic cancer cells was further investigated by subjecting nuclear extract preparations to gel filtration column chromatography analysis (Superdex200, exclusion limit 200 kD), as described in ‘‘Materials and Methods’’, in conjunction with immunoblotting analysis. The collected fractions, which showed peak absorbance at 280 nm (data not shown) were immuno- probed. Results showed that the EGFR protein first appears in the fraction 23 and is present together with Stat3 and Src, and the three proteins are concurrently detected in the fractions 23–29 (Fig. S1A). Results also showed detectable Stat3 and Src proteins in fractions 30 through 32 well after EGFR was completely eluted (Fig. S1A). Analysis of the previously-reported EGFR protein partner, RNA helicase A (RHA) [24] also showed detectable levels that were predominantly in the fractions 23 and 24 (Fig. S1A, RHA). These fractions were further subjected to co-immunopre- cipitation analysis to validate the presence of the complex. Immunoblotting analysis of EGFR immunecomplex prepared from the pooled fractions 23–27 further showed the presence of Src and Stat3 (Fig. S1B). The early and the concurrent elution of EGFR, Src and Stat3 raise the possibility that each of the proteins is part of a higher molecular weight protein complex, and also that the three proteins associate as part of the same complex, as suggested by the immunecomplex formation. There has been a previous report about nuclear EGFR/Stat3 complex under conditions of cellular stimulation by EGF [10]. We therefore investigated whether the nuclear EGFR, Src, Stat3 complex was present constitutively (without ligand stimulation) in tumor cells. Immunoblotting analysis did not detect any appreciable levels of Stat3 or Src in EGFR immunecomplex or of Src or EGFR in Stat3 immunoprecipitate from the nuclear extracts prepared from PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e19605