Western blot was performed as previously described [12 (link)]. Specific primary antibodies [mouse monoclonal anti-human β-actin (Sigma-Aldrich), anti-ASS1, anti-OTC, anti-PARP, (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA), anti-Bcl-2, anti-cleaved caspase-3, anti-cyclin A2, D3, E1, H, anti-CDK4 (Cell Signaling Technology, Danvers, Massachusetts, USA), anti-Ki67 (ImmunoWay Biotechnology Company, Texas, USA), anti-PEG (RevMAb, San Francisco, USA) antibodies] and corresponding horseradish peroxidase (HRP)-conjugated secondary (Cell Signaling Technology) were purchased. An enhanced chemiluminescence (ECL) kit (GE Healthcare) was used to detect protein expression. Beta-actin was selected as reference protein [14 (link)].
Immunohistochemistry and immunofluorescence staining were carried out according to standard protocol. Anti-cytokeratin 1 (Santa Cruz Biotechnology), anti-PEG and corresponding Alexa Fluor anti-mouse or anti-rabbit (Life Technology) antibody were used. The slides were mounted with Prolong® Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies). Photos were taken with Nikon Ni-U fluorescence microscope (Nikon, Tokyo, Japan) equipped with camera/detector Diagnostic Instrument RT3 Slider (Meyer Instruments, Houston, USA).
Immunohistochemistry and immunofluorescence staining were carried out according to standard protocol. Anti-cytokeratin 1 (Santa Cruz Biotechnology), anti-PEG and corresponding Alexa Fluor anti-mouse or anti-rabbit (Life Technology) antibody were used. The slides were mounted with Prolong® Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies). Photos were taken with Nikon Ni-U fluorescence microscope (Nikon, Tokyo, Japan) equipped with camera/detector Diagnostic Instrument RT3 Slider (Meyer Instruments, Houston, USA).