Epha2

MIA PaCa-2, PANC-1, BxPC-3, and AsPC-1 cells were all obtained from the American Type Culture Collection (ATCC). LT2 immortal normal mesenchymal cells were purchased from Millipore. MIA PaCa-2 and PANC-1 were maintained in DMEM plus 10% FBS. BxPC-3 and AsPC-1 cells were maintained in RPMI plus 10% FBS. LT2 cells were maintained with media according to distributor's instructions. Cell lines were expanded and cryopreserved at early passages and new vials were thawed and used for experiments approximately every 3 months. 5 × 10⁵ cells were plated in 6-cm dishes and treated as described. After 48 hours, whole cell lysates were prepared and western blotting analysis was carried out as previously described [37 (link)]. Primary antibodies used for these studies were EphA2 (1:1,000, Cell Signaling) and EF1-α (1:5,000, Sigma). Representative data are reported in Figure 2A.
Normal pancreatic tissue (N-pancreas) chronic pancreatitis tissue (CP), and Pancreatic ductal adenocarcinoma (PDAC) tissue were obtained from an IRB approved protocol at Cedar-Sinai Medical Center (34086) and probed for EphA2 expression using a rabbit anti-EphA2, (ab78002, abcam, 1/200). Representative data are reported in Figure 2B–2D.

Proteins were separated on an 8% SDS-PAGE and transferred onto a PVDF membrane. After blocking with 5% bovine serum albumin (BSA; Bio-Rad, CA, USA) for 1 h at room temperature, membranes were incubated with primary antibodies at 4 °C overnight. Antibodies against C1GALT1, GAPDH, Ephrin A1, and FAK were purchased from Santa Cruz Biotechnology. Antibodies against EGFR, p-EGFR, HER2, p-HER2, p-AKT, EPHA2, p-EPHA2, p-ERK, ERK, p-STAT3, STAT3, and p-FAK were purchased from Cell Signaling Technology (MA, USA). Antibodies against AKT, p-Src, and Src were purchased from GeneTex Inc. (CA, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies, and proteins were detected using ECL reagents (GE Healthcare Life Sciences).

Immunoblotting was performed as described^{2 (link)} with antibodies obtained from Cell Signaling Technologies: EphA1 (rabbit monoclonal D6V71), EphA2 (rabbit monoclonal D4A2), EphA3/A4/A5 (rabbit monoclonal D2C11), Erk1/2 (rabbit monoclonal 137F5), phospho-Erk1/2 (rabbit monoclonal 20G11), Stat1 (rabbit monoclonal 43H3), phospho-Stat1 (rabbit monoclonal D4A7), Stat3 (rabbit monoclonal D3Z2G), phospho-Stat3 (rabbit monoclonal D3A7) and cleaved Notch1 (rabbit monoclonal D3B8). Antibodies were used at a 1:1000 dilution of the supplied stock following the protocols specified by the supplier. Blots were developed using Thermo Fisher ECL Plus and visualized with a My ECL imager. Image acquisition time was adjusted to visualize all bands without saturating the brightest bands (from 30 seconds to 30 minutes depending on the efficacy of the antibody). Data were analyzed using Thermo My Image Analysis software, version 2.0. Images were inverted to display dark bands on a light background and in some cases brightness or contrast were adjusted to show lighter bands more distinctly, again without saturating the darkest bands. Images were then cropped, arranged and converted to TIFs with Photoshop, version CS6.

For IF of 3D organoids, organoids in Matrigel were resuspended in 500 μl cell recovery solution (Thermo Fisher Scientific, 12648–010) with a widened pipette, transferred to falcon tube and incubated for 45 min on ice until Matrigel was dissolved. For IF in 2D, dissociated gastric organoids were seeded onto 8 well μ-slides (IBIDI, 80826) to form 2D monolayers for immunofluorescence staining. Cells were grown for 7–14 d to reach approximately 90% confluency. Fixation was performed with 4% PFA for 20 min at room temperature, washed three times with PBS and permeabilized in 1 X PBS supplemented with 0.3% Triton-X, 1% DMSO and 1% fresh BSA for 1 h. Stainings were performed with primary antibodies (Occludin: Santa Cruz Biotechnology, sc-133256; CD45: Santa Cruz Biotechnology, sc-1178; EPHA2: Cell Signaling, 6997S; Pan-cytokeratin: Santa Cruz Biotechnology, sc-8018; E-cadherin: BD Bioscience, 610182) in 1 X PBS supplemented with 5% goat serum (Thermo Fisher Scientific, 31872) overnight at 4°C followed by Alexa Fluor (AF) 488 or AF647-conjugated secondary antibodies (Cell Signaling) in 1 X PBS supplemented with 5% goat serum for 3 h at room temperature. Actin filaments were stained with Phalloidin (Thermo Fisher Scientific, A22283) and DNA was stained with Hoechst 33342. After washing three times with PBS, stained cells were visualized using a confocal microscope (Leica, TC5 SP5 X).