Western blotting, immunofluorescent staining, and immunohistochemistry

HuH-7, HuH-7^R and Sk-Hep-1 cells were subjected to lentivirus-mediated knockdown, EphA2 (wild-type and mutant) expression, or treatment with Ephrin-A1-Fc (R&D) or prazosin (Sigma-Aldrich). All samples were lysed in lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, a 1× phosphatase inhibitor, and a 1× protease inhibitor cocktail (pH 7.4). The lysates were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. Antibodies for western blotting against EphA2, p-EphA2 Y772, p-EphA2 S897, cleaved caspase 3, and cleaved poly ADP-ribose polymerase (PARP) were acquired from Cell Signaling Technology, and an actin antibody was acquired from Millipore. Western blot analyses were conducted as described previously¹⁹. The ligand-dependent EphA2 internalization induced by prazosin was examined via immunofluorescent staining with a specific anti-EphA2 antibody, as described previously¹⁶. Briefly, HuH-7^R cells were seeded on coverslips overnight and then treated with 10 μM prazosin for 0, 2, and 4 h at 37 °C. After treatment, the cells were fixed, blocked, and immunostained with an antibody against EphA2 followed by a tetramethylrhodamine-isothiocyanate (TRITC)-labeled secondary antibody. Nuclear staining was performed with 4,6-diamidino-2-phenylindole (DAPI), and images were captured using a Carl Zeiss LSM880 confocal microscope (Zeiss, Jena, Germany). Apoptosis induced by EphA2 inhibition was analyzed via Hoechst 33342 staining²⁰. The percentage of apoptotic cells in the groups treated with 10 μM prazosin for 0, 2, 4, or 6 h was determined by counting the cells showing nuclear fragmentation. Xenograft tumors harvested from in vivo animal experiments were examined for the expression of EphA2, p-EphA2 S897, Ki-67, and cleaved caspase-3 via immunohistochemical analyses as described previously¹⁹.