Mab3496

ZBTB7A stable overexpression cells were seeded into a 6-well plate. The next day, cells were trypsinized and collected through centrifugation. The cell pellets were resuspended with 100 μL PBS and lysed with 100 μL 2×Laemmli sample buffer (Bio-Rad, 1610737). The cell lysate was heated at 95°C for 5 min and quantified by Bradford assay (Pierce, 23246). 30 μg of cell lysate was loaded onto 4–20% Mini-PROTEAN TGX Stain-Free Protein gels (Bio-Rad, 4568094) and run at 100 V for 1 h. The gel was transferred to 0.45 μm nitrocellulose membrane at 60 V for 1 h and the membrane was further blocked with 5% milk diluted with PBST for 3 h. After three washes with PBST, recombinant anti-ZBTB7A antibody (R&D, mab3496) at a 1:1000 dilution, anti-alpha tubulin antibody (Abcam, ab179484) or anti-alpha actin antibody (Santa Cruz, sc-47778) at a 1:1000 dilution was incubated overnight at 4°C. After three washes with PBST, goat anti-rabbit-HRP antibody (Life Technologies, A16104) or goat anti-armenian hamster-HRP antibody (Novus, NB100-2066) was incubated with the membrane for 1 h. Finally, Clarity Western ECL Substrate (Bio-Rad, 1705060) was used for imaging with Amersham Hyperfilm ECL (GE Healthcare 28906839).