Analyzing EphA2 Expression in Pancreatic Cancer

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.