Epha2 ps897

For immunostaining or western blotting, primary antibodies against the following proteins were used: MYOF (Santacruz); EGFR, pEGFR, EPHA2, and EPHA2-pS897 (Cell Signaling); VIM and E-cadherin (Abcam); and actin (Pierce). For western blot analysis, cells were washed once with ice-cold PBS and then lysed in cold RIPA buffer supplemented with protease inhibitor cocktail (Roche Applied Biosciences). Cell lysate was centrifuged at 12,000 × g for 30 min, boiled in 2 × loading sample buffer, separated on a 10% gradient SDS–PAGE gel and blotted onto a PVDF membrane. The membrane was blocked with 0.1% Tween-20 in TBS containing 5% non-fat milk and probed with the indicated primary antibody, followed by incubation with the appropriate secondary antibody conjugated to horseradish peroxidase (Sigma), and the signals were visualized via ECL (Millipore).

Briefly, the samples were fixed with 4% formaldehyde for 48 h at room temperature and embedded in paraffin. Then, the paraffin-embedded tissue sections at 5-8 μm thickness were transferred onto glass slides. The slides were deparaffinized, rehydrated, and pressure cooker heated for 2-5 min in EDTA for antigen retrieval. Endogenous peroxidase activity was inactivated by adding an endogenous peroxidase blocker (OriGene, Beijing, China) for 15 min at room temperature. Slides were incubated overnight at 4°C with KLF5 (Proteintech, 66850-1-Ig), EphA2 (Santa Cruz, sc-398832), and EphA2 pS897 (Cell Signaling, #6347) antibodies. Next, the slides were washed three times with 1×PBS and incubated with secondary antibody (OriGene, Beijing, China) at room temperature for 20 min, DAB concentrate chromogenic solution (1:200 dilution of concentrated DAB chromogenic solution), counterstained with 0.5% hematoxylin, dehydrated with graded concentrations of ethanol for 3 min each (70%-95%-100%), and finally stained with dimethyl benzene. Immunostained slides were evaluated by light microscopy. IOD score: integrated optical density score.