Pi3k 4292

Total cellular protein was lysed using the RIPA buffer (Cell Signal Technology, Inc., MA, USA) and quantified by a BCA kit (Beyotime Biotechnology, Shanghai, China) following the principle of manufacturers’ protocols. Then 20 µg of the total protein was separated with 10% SDS-PAGE and then transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was blocked by pre-incubation with 5% skim milk for 30 min at room temperature and then incubated with the primary antibodies, including RPN1 (12894-1-AP, 1:500, Proteintech), PI3K (4292, 1:1,000, Cell Signaling), AKT (ab179463, 1:2,000, Abcam), mTOR (ab2732, 1:3000, Abcam), p-PI3K (ab182651, 1:500, Abcam), p-AKT (9271, 1:1,000, Cell Signaling), p-mTOR (2974, 1:1,000, Cell Signaling) and GAPDH (#2118, 1:1,000, Cell Signaling) at 4 °C overnight. On the next day, after three washes, the membranes were incubated with corresponding secondary antibodies, including goat anti-mouse IgG-HRP (sc-2005, Santa Cruz) and goat anti-rabbit IgG-HRP (sc-2004, Santa Cruz) diluted at 1:5,000 for 30 min at room temperature. The target protein was detected using Pierce™ ECL Western (Thermo Scientific, 32,209) and photographed by the chemiluminescence imaging analysis system (Bio-Rad, USA). The relative gray value was quantified by Image J (NIH, USA). GAPDH was used as the internal control [20 (link), 21 (link)].