Rabbit anti nrf2 d1z9c

To assess the NRF2 level, 25 µg of protein isolated from C2C12 and C2C12-NRF2 cells were loaded onto a 12% SDS-PAGE gel. After electrophoresis (100 V), dry transfer to the nitrocellulose membrane was performed using the iBlot Dry Blotting System (Invitrogen, Waltham, MA, USA). The membranes were then blocked with 5% milk in Tris-buffered saline (TBS) for 1 h at room temperature (RT). Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies rabbit anti-NRF2 (D1Z9C; Cell Signaling Technology, Danvers, MA, USA; molecular weight ~100 kDa) and mouse anti-α-tubulin (DM1A; Cell Signaling Technology; molecular weight ~52 kDa) diluted 1:100 or 1:1000 in blocking solution, respectively. The next day, the membranes were washed 3 times (5 min each) with TBS + 0.1% Tween 20 and then secondary antibodies conjugated with HRP, goat anti-rabbit (1:1000; Cell Signaling Technology) and goat anti-mouse (1:10,000; BD Pharmingen, San Diego, CA, USA ), were applied for 1 h at RT. The membranes were washed 4 times with TBS and incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) for 10 min in the dark and finally developed manually on the X-ray film. PageRuler Prestained (Thermo Fisher Scientific) protein ladder was used as a marker.