Ecl hrp detection kit

Total proteins were extracted from GBM cells using RIPA lysis buffer. Protein samples were quantified using a bicinchoninic acid protein assay kit (Beyotime). Equal amount of total protein was separated by SDS‐PAGE and then transfected onto a PVDF membrane. After incubation with 5% skim milk overnight, the membrane was incubated with antibodies against β‐catenin (1:500; Abcam), Axin2 (1:500; Abcam), c‐myc (1:1,000; Abcam), cyclin D1 (1:1,000; Abcam), and GAPDH (1:1,000; Abcam), followed by incubation with HRP‐conjugated secondary antibody (1:1,000; Beyotime). Finally, the target proteins were visualized with a ECL‐HRP detection kit (Millipore).

Total protein was extracted using super RIPA lysis buffer and then quantified by a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Twenty‐five micrograms of protein was loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (PVDF). After being blocked with 5% nonfat milk overnight at 4°C, the PVDF membrane was incubated with primary antibody for 2 hr at room temperature and then with horseradish peroxidase (HRP)‐conjugated secondary antibody (1:1,000; Beyotime, China) for another 1 hr at 37°C. Primary antibodies included antibodies against USP44 (1:500; Abcam), cleaved‐caspase3 (1:500; Abcam), proliferating cell nuclear antigen (PCNA; 1:500; Abcam), β‐catenin (1:1,000; CST), Axin1 (1:500; Abcam), c‐myc (1:1,000; Abcam), cyclin D1 (1:1,000; Abcam) and β‐actin (1:1,000; CST). Immunodetection was conducted by an ECL‐HRP detection kit (Millipore).