Tumor Protein Analysis by Western Blot

Following the experiment, mice were euthanized with an overdose of anesthetic combined with carbon dioxide. Tumors were quickly obtained from the mice, then coarsely minced and homogenized. Western blotting was then performed as previously described [98 (link),99 (link),100 (link)]. For detection, we used antibodies against β-actin, Bcl-2, and COX-2 from Sigma-Aldrich Inc. (St. Louis, MO, USA); VEGF and EGFR were also purchased from Sigma-Aldrich (St. Louis, MO, USA); and cleaved caspase 3, phosphorylated ERK (threonine 202/tyrosine 204), and ERK were from Cell Signaling Technology (Beverly, MA, USA). Enhanced chemiluminescence reagents (Thermo Scientific, Rockford, MA, USA) generated the immunoreactive signals and UVP ChemStudio (Analytik Jena, Upland, CA, USA) was used for signal detection. The quantification of protein expression and phosphorylation was performed using ImageJ software from the National Institutes of Health (NIH; Bethesda, MA, USA).