Goat anti na k atpase

Total protein (20–30 µg) was resolved in 4–15% Tris-Glycine SDS-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA), and then transferred to a nitrocellulose or PVDF membrane. Membranes were blocked by TBST (Tris-buffered saline with 0.1% Tween-20) containing 5% BSA at 22 °C for 1 h. Primary antibodies were diluted in 1% BSA-TBST as follows: 1:10,000 for rabbit anti-Fpn (Abcam, ab85370) and mouse anti-APP (Millipore, 22C11); 1:5000 for mouse anti-Hp (Abcam, ab56729), rabbit anti-β-actin (Cell Signaling, 13E5), and goat anti-Na–K-ATPase (Santa Cruz Biotechnology, C16), then incubated with membranes at 4 °C overnight. After wash, membranes were incubated for 1 h at 22 °C with the cognate HRP-conjugated secondary goat anti-mouse, goat anti-rabbit, or donkey anti-goat antibody diluted in TBST containing 3% BSA (Santa Cruz Biotechnology). The immunoblot was developed using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Waltham, MA) and band intensity was quantified in Image Studio Lite (Licor Biosciences, Lincoln, NE).