Chemi lumi one l western blotting substrate

Western blotting was performed as described previously.^{40 (link)} To isolate nuclear fractions, cells were treated with a hypotonic buffer for 5 min on ice. Proteins were separated by sodium-lauryl-sulfate-polyacrylamide gel electrophoresis and transferred electro-phoretically onto PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% (W/v) phospho-blocker (Cell Biolabs. Inc., San Diego, CA, USA) for 1 h and incubated with primary antibodies such as anti-β-actin (A2066, Sigma, St Louis, MO, USA), anti-phospho-AKT-Ser473 (4060, Cell Signaling, Beverly, MA, USA), anti-AKT (4685, Cell Signaling), anti-cyclin D1 (Nichirei Bioscience), for 1 h at room temperature or overnight at 4 °C. Membranes were then incubated for 1 h at room temperature with the secondary antibody of HRP-conjugated goat anti-rabbit IgG (GE Healthcare, Buckinghamshire, UK) or HRP-conjugated goat anti-mouse IgG (R&D Systems, Minneapolis, MN, USA). The protein bands were visualized with Chemi-Lumi One L Western blotting substrate (NacalaiTesque). Band intensity was measured by densitometry using the Image Lab Software (Bio-Rad).