Western Blotting of GSC Proteins

Western blotting was performed as previously described [23 (link)]. Briefly, the total proteins of GSCs or tissues were isolated using a total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China). Protein lysates were prepared, subjected to SDS-PAGE, transferred onto polyvinylidene difluoride membranes and blocked with 2% bovine serum albumin (KeyGen Biotechnology). The primary antibodies against ISL2 (1:1000; Abcam), VEGFA (1:1000; Abcam), VEGFR2 (1:1000; Abcam), p-VEGFR2 (1:1000; Abcam), MEK1/2 (1:1000; Abcam), p-MEK1/2 (1:1000; Abcam), ERK1/2 (1:500; Abcam), p-ERK1/2 (1:500; Abcam), and β-actin (1:2000; Proteintech, Rosemont, IL, USA) were incubated at 4 °C overnight. After secondary antibody (Proteintech) incubation, the bands were detected using a chemiluminescence ECL kit (Beyotime Biotechnology, Beijing, China) and quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).