Irdye 680rd goat anti mouse igg h

The RIPA lysis buffer (Cat No. P0013B, Beyotime Institute of Biotechnology, Jiangsu, China) was enriched with a protease cocktail inhibitor (Cat No. 4693132001, Roche) that was employed to isolate total proteins of the cells. The quantities of the isolated proteins were determined using the BCA protein assay kit (Cat No. P0009, Beyotime Institute of Biotechnology, Jiangsu, China). Thereafter, 20 μg of total proteins was fractionated on a 12% SDS-PAGE gel and transfer embedded onto 0.22 μM PVDF membranes (Cat No. ISEQ00010, EMD Millipore). Subsequently, the membranes were blocked with 5% nonfat milk at room temperature (RT) for two hours and then incubated overnight at 4°C overnight with the following specific primary antibodies: anti-NMU (1 : 200), anti-p-Erk1/2(1 : 1000), anti-Erk1/2(1 : 1000), anti-p-FoxO3 (1 : 1000), anti-FoxO3 (1 : 1000), anti-cyclin A2 (1 : 1000), anti-cyclin B1 (1 : 1000), anti-cyclin D1 (1 : 1000), anti-P21 (1 : 1000), anti-P27 (1 : 1000), and anti-beta-actin (1 : 5,000).After that, the membranes were incubated with the corresponding IRDye® 800CW Goat anti-Rabbit IgG (H + L) (1 : 15,000; Cat No. 926-32211; LI-COR) or IRDye® 680RD Goat anti-Mouse IgG (H + L) (1 : 15,000; Cat No. 926-68070; LI-COR) at room temperature (RT) for a further one hour. An Odyssey® CLx imaging system (LI-COR) was employed to capture blot images.

Mouse hippocampus protein lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently immunoblotted onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk at room temperature for 1 h. After TBST washing (three times, 5 min per wash), the membranes were incubated with the indicated primary antibodies at 4 °C overnight with shaking. The primary antibodies included β-actin (Sigma-Aldrich, A5316), lamin B1 (HUABIO Antibodies, ET1606-27), HIF-1α (Abcam, ab228649), HIF-2α (Novus, NB100-122), Acer2 (PA5-39016), PP2A (Invitrogen, MA5-32920), p-PP2A (Invitrogen, MA5-32158), α-syn (BD, 8249518), and p-α-syn (Abcam, ab51253). After incubation, the membranes were washed three times and then incubated at room temperature for 1 h with secondary antibodies, including IRDye 680RD goat anti-mouse IgG (H + L) (Licor, 926-68070), IRDye 680RD goat anti-rabbit IgG (H + L) (Licor, 926-68071), IRDye 800CW goat anti-mouse IgG (H + L) (Licor, 926-32210), IRDye 800CW goat anti-rabbit IgG (H + L) (Licor, 926-32211). Membranes were scanned using a detection system (Odyssey, USA), and band intensities were normalized to β-actin. Statistical analyses were performed using ImageJ and GraphPad software.