Mouse anti e cadherin

Cells were grown in chamber slides (Nalge Nunc International, Rochester, NY, USA). They were fixed for 15 min with 4% paraformaldehyde and washed several times with PBS. Coverslips were incubated in blocking solution containing 2% BSA in PBS for 1 hr, and incubated with the appropriate primary antibodies {anti-mouse E-cadherin and anti-rabbit β-catenin antibodies (Santa Cruz Biotech, CA, USA)} for 1 hr at room temperature. After incubations with appropriate secondary antibodies (E-cadherin, goat anti-mouse IgG-FITC; β-catenin, goat anti-rabbit rhodamine-conjugated IgG: Santa Cruz Biotech, CA, USA), fluorescence was visualized by epifluorescence con-focal microscopy (Pascal, BIORAD, (Hercules, CA, USA).

After the specific time of incubation, MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were fixed in p-formaldehyde (4% v/v in PBS) for 5 minutes. The cells were permeabilized in Triton X-100 (0.5% v/v in PBS) for 5 minutes, and then incubated in goat serum (20% v/v PBS) for 30 minutes, and with rabbit anti-ANXA1 antibody (1:100; Invitrogen), mouse anti-FAK (1:100; BD Transduction Laboratories), mouse anti-E-cadherin (1:250; Santa Cruz Biotechnology) and/or mouse anti-vimentin (1:500; Santa Cruz Biotechnology) overnight at 4°C. After two washing steps with PBS, cells were incubated with anti-rabbit and/or anti-mouse AlexaFluor (488 and/or 555; 1:1000; Molecular Probes) for 2 hours at RT and then with FITC-conjugated anti-F-actin (5 μg/ml; Phalloidin-FITC, Sigma) for 30 minutes at RT in the dark. The coverslips were mounted in glycerol (40% v/v PBS). A Zeiss LSM 710 Laser Scanning Microscope (Carl Zeiss MicroImaging GmbH) was used for data acquisition. To detect nucleus, samples were excited with a 458 nm Ar laser. A 555 nm He-Ne laser was used to detect emission signals from ANXA1 stain. Samples were vertically scanned from the bottom of the coverslip with a total depth of 5 mm and a 63× (1.40 NA) Plan-Apochromat oil-immersion objective. Images were generated with Zeiss ZEN Confocal Software (Carl Zeiss MicroImaging GmbH).

Glass slides prepared as mentioned above. The cells were incubated with mouse anti-E-cadherin and anti-β-catenin (1 : 50; Santa Cruz) antibodies at 4 °C overnight followed by washing with PBS three times. Coverslips were then incubated with Texas Red-conjugated anti-mouse antibodies (1 : 200; Invitrogen) for 30 min at room temperature, then stained with 6-diamidino-2-phenylindole (1 : 10 000; Invitrogen).

Protein was collected and extracted from A549 and H1299 cells with RIPA lysis buffer and protease inhibitor cocktail and protein phosphatase inhibitor. The samples were then transferred to PVDF membranes, following electrophoresis and the membranes were incubated with rabbit-anti SMAD3 (Cell Signaling Technology, Inc.) or mouse-anti E-cadherin or mouse-anti N-cadherin (both Santa Cruz Biotechnology, Inc.) primary antibodies(SC-8426 for E-cadherin, SC-8424 for N-cadherin) overnight at 4 °C. The following day, the membranes were incubated with indicated secondary antibodies (Santa Cruz Biotechnology, Inc, SC-2005) for 1 h at room temperature. Detection was performed using the electrochemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). β-actin (Santa Cruz Biotechnology, Inc.) was used as the internal control.

For immunofluorescent staining, HONE1 or 5-8F cells grown on the surface of cover slides were transfected with LV-con and LV-Klf4 separately, cells were fixed with 4% paraformaldehyde, rehydrated, and incubated with mouse anti-E-cadherin, anti-N-cadherin (Santa Cruz) and anti-Vimentin (Cell Signaling Technology, Danvers, MA) at room temperature for 40 min. Subsequently, cells were incubated with Alexa Fluor 594-conjugated anti-mouse antibody (Molecular Probes; Invitrogen Corp.) for 40 min at room temperature. The nuclei were stained with DAPI. Slides were examined with a fluorescent confocal microscope (Olympus FV1000, Japan), as our previously fully described [55 (link)].