The complexes were then precipitated, washed, eluted with lamelli buffer and then subjected to SDS-PAGE and Western blotting analysis probing for Stat3. Immunostaining with laser-scanning confocal imaging Panc-1 cells growing on coverslips in 12-well plates were treated with or without inhibitors for 1 or 24 h and subjected to immunostaining and fluorescence or laser-scanning confocal microscopy, as previously described [21]. Cells were fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, permeabilized with 0.25% Triton X-100 for 10 min, and washed three times with PBS. Specimens were then blocked in 0.1% BSA in PBST for 30 min and incubated overnight at 4uC with rat monoclonal anti-EGFR (Santa Cruz), mouse monoclonal anti-Src (Cell Signaling), and rabbit polyclonal anti-Stat3 (Cell Signaling) antibodies at 1:50 dilution (in 0.1% BSA). Subsequently, cells were rinsed three times in PBST, incubated for 1 h at room temperature in the dark with 1:1000 dilutions of the three AlexFluor secondary antibodies, ALexaFLuor405 (goat anti- mouse), AlexaFluor488 (donkey anti-rabbit) and AlexaFluor546 (goat anti-rat) (Molecular Probes, Invitrogen) for EGFR, Src and Stat3 detection, respectively. Specimens were then washed three with PBST. Subsequently, coverslips were removed and mounted on slides using Fluoromount-G (Southern Biotech, Birmingham, AL) and prevented from drying by sealing the edges with nail paint. Slides were stored in the dark at 4uC until images were captured. For negative staining, secondary antibodies were added without the primary antibodies. Confocal analysis was performed by the examination of slides under Leica TCS SP5 confocal microscope (Germany) at appropriate wavelengths. Images were captured and processed using the Leica TCS SP 5 software. Chromatin Immunoprecipitation (ChIP) and Sequential ChIP Analyses For ChIP assay, cells in culture were treated with formaldehyde at a final concentration of 1%, for 10 min at room temperature followed by treatment with glycine at a final concentration of 0.125 M for 5 min at room temperature for cross-linking. Subsequently, cells were washed with ice-cold PBS and resuspended in and lysed with lysis buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM dithiothreitol), 1X TLA, 1 mM phenylmethylsulfonyl fluoride, 5% Nonidet P-40, and centrifuged. Then nuclear pellet was resuspended in nuclei lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS and protease inhibitors) (Roche, Indianapolis, IN). The nuclear lysates were sonicated (Omni International, Kennesaw, GA) at 30% power for 3 pulses for 10 s intervals on ice to shear DNA. The chromatin solution was pre-cleared with protein A/G agarose beads (Santa Cruz) for 1 h at 4uC with rocking. Then the pre-cleared lysates were immunoprecipitated by incubating with anti-EGFR, anti-Src, or anti- Stat3 antibodies or with IgG (for no antibody) (Santa Cruz) at 4uC overnight with rocking. The complexes were collected with 20 mL protein A/G agarose beads (Santa Cruz), washed three times using wash buffer A (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0) and two times with wash buffer B (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl, pH 8.0). Then complexes were eluted with freshly prepared elution buffer (1% SDS, 100 mM NaHCO3). Cross-links were reversed by heating at 65uC in the presence of NaCl followed by proteinase K treatment (20 ml of a 20 mg/ml) for 6 h. The DNA was recovered and purified using DNA purification kit from Qiagen (Valencia, CA). The purified chromatin immunoprecipitated DNA was next used as a template for the polymerase chain reaction (PCR) amplification of the c-Myc promoter using the primers, Forward, 59AAAAGGGGAAA- A Functional Nuclear EGFR, Src and Stat3 Complex GAGGACCTGG-39, and Reverse, 59-TAAAAGGGGCAAGTG- GAGAGC-39 or the TWIST promoter using the primers, Forward, 59- AGTCTCCTCCGACCGCTTCCTG -39. Reverse: 59- CTCCGTGCAGGCGGAAAGTTTGG -39 (In- vitrogen). The PCR products, 133 bp for c-Myc and the 332-bp for TWIST [22] were resolved on 2% agarose gel. The sequential ChIP studies were performed as previously reported [10] and following the sequential immunoprecipitation studies described, using the first primary antibody (anti-EGFR) and the second primary antibody (anti-Src). The recovered complexes after the secondary immunoprecipitation were eluted with the elution buffer and then subjected to ChIP assay, as described. Gel filtration chromatography The pre-packed Superdex 200 10/30 GL glass column was purchased from GE Healthcare Life Sciences (Piscataway, NJ). The chromatography system used in the study was the BioLogic Duoflow System (Bio-Rad, Hercules, CA). The chromatography analysis was performed following the manufacturer’s instructions with a general run sequence of equilibration, load, elution, regeneration and storage. RIPA Buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Nonidet P-40) was used as the mobile phase buffer. Samples (Panc-1 cell lysate, 2 mg total protein in a volume of 250 ml) were loaded unto the column, then flow rate was adjusted to 0.25 ml/min, and then fraction (500 ml) collection was initiated right after the sample loading and monitored by the eluent absorbance at 280 nm. According to the absorbance peaks, fractions 21–34 were selected and subjected to immunoblotting analysis using antibodies against EGFR, Stat3, and Src (Cell Signaling), and against RNA helicase A (RHA) (Abcam, Cambridge, MA). For immunoprecipitation assay, 100 ml each of fractions 23–27 were pooled, from which EGFR immunecomplex was precipitated using anti-EGFR antibody (Cell Signaling) and subjected to immunoblotting analysis for Stat3, Src and EGFR. Preparation of anti-EGFR and mouse IgG1-GNP probes Gold nanoparticles (GNPs), 0.1 nM, with a diameter of 40 nm were purchased from Ted Pella Inc. (Redding, CA). Mouse monoclonal anti-EGFR [F4] antibody was purchased from Abcam (cat. no. ab62, conc. 1.2 mg/ml), and non-specific mouse mono- clonal IgG1 was purchased from Sigma (cat. no. M9629, conc. 1 mg/ml). Polyclonal anti-Stat3 (conc. 0.2 mg/ml), polyclonal anti- Src (0.1 mg/ml), polyclonal anti-EGFR (0.2 mg/ml) and non- specific rabbit IgG (0.4 mg/ml) were purchased from Santa Cruz. All other chemicals and buffer ingredients for the assay development were purchased from Sigma. The anti-EGFR-GNP probe was prepared by adding 10 ml of mouse monoclonal anti-EGFR antibody to 1 ml of GNPs. After incubation for 15 min at room temperature, the probe was blocked with 2.5 mg BSA for 30 min. After the centrifugation at 10,000 rpm for 5 min, the supernatant was discarded and the nanoparticle residue was redispersed in 0.5 ml of 0.25% BSA in 10 mM phosphate buffer (PB). The probe was then used in the assay. The negative control mouse IgG1-GNP probe was prepared by adding 10 ml of mouse monoclonal IgG1 antibody to 1 ml GNPs, and following the procedure identical to the one used for the EGFR probe preparation. Mouse IgG1 was used here to prepare the control probe, because the anti-EGFR monoclonal antibody is a mouse IgG1 type antibody. Detection and kinetic binding study of EGFR from nuclear extract to the GNP probes The Panc-1 nuclear extract sample was diluted in phosphate buffer (PB) to 1 mg/ml of total protein. In a sample cell for PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e19605