A Functional Nuclear EGFR, Src and Stat3 Complex experiment (data not shown). This result indirectly suggested that the EGFR protein detected by the nanoparticle immunoprobe from the nuclear extract exists as a complex with Stat3 and Src protein. Detection of EGFR, Src and Stat3 heterocomplex by immunofluorescence with laser-scanning confocal microscopy Immunofluorescence with laser-scanning confocal microscopy allowed visualization of the intracellular distribution and localiza- tion patterns of EGFR, Src and Stat3. Immunofluorescence with laser-scanning confocal microscopy confirmed the localization patterns and showed that in contrast to the negative staining (Fig. 5B(i), left panel), EGFR appearance is punctuate (red) at the plasma membrane, and in the cytoplasm and the nucleus (Fig. 5B(i), single). Similar localizations for Src (blue) and Stat3 (green) were observed, but with greater presence in the nucleus (Fig. 5B(i), single). There are stainings for colocalization of EGFR and Src (magenta, see arrows), EGFR and Stat3 (yellow, see arrows), Src and Stat3 (cyan, see arrows), and of all three entities (pale yellow/white, see white arrows) at the plasma membrane, cytoplasm, perinuclear and nuclear spaces (Fig. 5B(i), merge). Figure 5. Immunofluorescence with laser-scanning confocal microscopy of EGFR, Src and Stat3 association in HPDEC or Panc-1 cells. Cultured normal human pancreatic duct epithelial cells (HPDEC) (A) or pancreatic cancer, Panc-1 cells (B) were fixed, stained with primary antibodies against EGFR, Src and Stat3 and their corresponding secondary antibodies, ALexaFLuor405 (goat anti-mouse, EGFR, red), AlexaFluor488 (donkey anti-rabbit, Src, blue) and AlexaFluor546 (goat anti-rat, Stat3, green) and analyzed by laser-scanning confocal microscopy for localization (single) and colocalization (merge) studies of EGFR (red), Src (blue) and Stat3 (green) and the effects of treatment (i) without or (ii) with ZD1839 (ZD) or (iii) Dasatinib (Das) for the indicated times. Confocal images were collected using Leica TCS SP5 microscopes; Cyan, magenta, yellow and white/ pale yellow arrows denote merged colors; single, one color capture, merged, three-color capture. Data are representative of 3 independent studies. doi:10.1371/journal.pone.0019605.g005 PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e19605