A Functional Nuclear EGFR, Src and Stat3 Complex Figure 1. Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 association in Panc-1 and Colo-357 cells. Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), and Stat3 (IP:Stat3), or of non-specific IgG non-immunoprecipitate prepared from whole-cell lysates of Panc-1 or Colo-357 cells untransfected (A and B) or transfected with EGFR siRNA, Src siRNA, or control (con) siRNA (C) and probing for Src, Stat3 and EGFR in the absence (A and C) or presence (B) of Stat3 blocking peptide (Stat3 BP), Src blocking peptide (Src BP) or EGFR blocking peptide (EGFR BP). 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 used in the assay; Data are representative of 3 independent studies. doi:10.1371/journal.pone.0019605.g001 human breast cancer, MDA-MB-231 and the human non-small cell lung cancer, A549 lines (Fig. S1C), suggesting a minimal possibility of the existence of a constitutive nuclear EGFR, Src, Stat3 complex in the two cancer cell types. These studies together show for the first time that EGFR, Src and Stat3 form a heteromeric complex in the nucleus of pancreatic cancer cells. Given the detection of the EGFR, Src and Stat3 complex in whole-cell and nuclear lysates, we were interested to determine the relative levels and the sizes of EGFR, Src and Stat3 in the different sub-cellular fractions. Membrane and cytosolic fractions, and nuclear extracts were prepared from Panc-1 cells according to established protocols and which involved using 10% Nonidet P-40 lysis, and a low-salt HEPES buffer extraction for cytosolic extract, a high-salt HEPES buffer extraction for nuclear extracts (18, 22), and 0.5% SDS buffer for the membrane fraction. Immunoblotting analysis of samples of equal total protein from the sub-cellular fractions shows that with respect to each of the EGFR, Src, or Stat3 protein, the size is the same in the membrane (Mem), cytosolic (Cyto), or nuclear fraction (Nuc) (Fig. 2C). Results further show that the level of the total EGFR protein is highest in the membrane, and is higher in the cytosolic fraction than in the nuclear extract (Fig. 2C (i)). By contrast, results show that Stat3 levels are highest in the cytosolic fraction, and higher in the nuclear extract, compared to the levels associated with the cell membrane (Fig. 2C(ii)). The results for Src also showed noticeable differences, with the membrane-associated levels higher than both the cytosolic and nuclear levels, which were nearly identical (Fig. 2C(iii)). Significantly, the non-denaturing conditions (10% Nonidet P-40, low- or high-salt extraction) for preparing the cytosolic and nuclear extracts raises the potential that the EGFR protein detected in the cytosolic compartment and in the nucleus is a soluble form, and further that the Stat3 protein in the cytosolic fraction and in the nucleus is potentially a dimer. Moreover, this is the first report of the nuclear presence of c-Src tyrosine kinase in cancer cells. The role of EGFR and Src tyrosine kinases in the nuclear EGFR, Src and Stat3 heterocomplex formation We determined the importance of EGFR or Src in the assembly of the nuclear complex by examining the effect of siRNA knockdown. Immunoblotting analysis of immunecomplexes of Stat3 (IP:Stat3), EGFR (IP:EGFR) or Src (IP:Src) prepared from nuclear extracts showed that the siRNA knockdown of Src (Fig. 3A(i), upper panel) has little effect on the association of Stat3 with EGFR in the nucleus (Fig. 3A(i), IP:Stat3, IP:EGFR), while similarly, the knockdown of EGFR (Fig. 3A(ii), upper panel) did not alter the binding of Stat3 to Src (Fig. 3A(ii), IP:Stat3, IP:Src). Scrambled (con) siRNA has no effect. Similar to the results for the PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e19605