Ab6721

The proteins of glioma cells and tissues were extracted using radio-immunoprecipitation assay (Sigma-Aldrich) and denatured at 100°C before they were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. After transferring to the polyvinylidene fluoride (Sigma-Aldrich) membranes and immersing in 5% skimmed milk, the membranes were conjugated with primary antibodies against Wnt7B (ab94915, 1:1,000, Abcam, Cambridge, MA, USA), Total-caspase 3 (ab32351, 1:5,000), cleaved-caspase 3 (ab32042, 1:5,000), N-cadherin (ab76057, 1:1,500), E-cadherin (ab1416, 1:100), fibronectin (FN, ab45688, 1:1,000), and snail (ab180714, 1:1,000), proliferating cell nuclear antigen (PCNA) (ab18197, 1:2,000), or GAPDH (ab8245, 1:2,000) at 4°C overnight. Following, the membranes were mixed with corresponding secondary antibodies (ab6721, 1:20,000; ab6789, 1:15,000) for 1 h. Finally, an enhanced chemiluminescence Western blotting system (Bio-Rad, Hercules, CA, USA) was used to detect these membranes.

0.5 ml, 4 °C Cell lysis buffer IP (Beyotime, China) containing protease and phosphatase inhibitor cocktail was used to lyse cells (3 × 10⁵) and tissues (1 mg, grinded) at 0 °C for 2 h to extract total protein. We applied centrifugation at 4 °C, 15000 r/min, collected the supernatant, trimmed the protein system and denatured at 95 °C for 5 min. Proteins were separated by 10% SDS-PAGE electrophoresis and transferred to PVDF (0.45 μm, Thermo Scientific) membrane. 5% skim milk was used to block membrane for 2 h. The relevant first antibodies were used to incubate PVDF membrane at 4 °C overnight; information on antibodies is as follows: anti-UCHL5 (1:1000 diluted, 38KDa, Abcam, ab133508); anti-NFRKB (1:500 diluted, 139KDa, Abcam, ab86154); anti-Ubiquitin (1:2000 diluted, Abcam, ab7780); anti-β-Actin (1:2000 diluted, 42 kDa, abcam, ab8226). The relevant second rabbit anti-mouse or anti-rabbit antibody (1:2000 diluted, abcam, ab6728 or ab6721) incubated strip at room temperature for 1 h. ECL detection reagent and Bio-Rad GelDoc XR were used to detect the intensity of the strip signal.