Ab32374

Mouse tissues and cell lysates were extracted using RIPA lysis buffer (20–188, Millipore) according to the manufacturer’s protocol. Cell culture supernatant was collected and concentrated with Amicon Ultra-15 tubes (10 KDa, Millipore). The following antibodies were used: anti-RRBP1 (PA5-21,392, Invitrogen), anti-RRBP1 (Ab95983, Abcam), anti-RRBP1 (HPA011924, Sigma), anti-Hsp70 (DF2698, Affinity), anti-β-actin (GTX109697, Genetex), anti-renin (H0005972-M01, Abnova), anti-ACE (MA5-32,741, Invitrogen), anti-β-tubulin (tcaba2, Taiclone), anti-SGK1 (ab32374, Abcam), anti-ADCY5/6 (PA5-75,274, Abnova), and anti-calnexin (ab22595, Abcam). The blots were detected with Trident pico Western HRP Substrate (GTX17435, Genetex), and images were analyzed using a UVP BioSpectrum Imaging System.

Total protein isolated from cells and tissues was lysed in 100 µL of lysis buffer at 4 °C for 30 min, followed by 20 min of centrifugation at 12,000 r/min, 4 °C to collect the supernatant. The protein concentration measurement was realized with a bicinchoninic acid kit (20201ES76, Yeasen Biotech Co., Ltd., Shanghai, China). Following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the isolated proteins were loaded onto a nitrocellulose filter membrane. The protein-carried membrane was blocked with 5% skimmed milk powder at 4 °C overnight and further probed with specific primary antibodies at 4 °C overnight, including rabbit anti-mouse antibodies to PDCD5 (ab75430, 1: 1000, Abcam Inc., Cambridge, UK), HDAC3 (ab32369, 1: 1000, Abcam), SGK1 (ab32374, 1: 500, Abcam), and CD3 (ab16669, 1: 25, Abcam). The membrane was re-probed with diluted horseradish peroxidase-marked goat anti-rabbit immunoglobulin G (IgG) antibody (ab6721, 1: 5000, Abcam) at ambient temperature for 1 h. After visualization, development, and photographic fixing, gray values of protein bands were analyzed by Quantity One software followed by quantitative protein analysis with GAPDH (ab181602, 1: 5000, Abcam) as the internal reference.

Nuclear and cytosolic fractions of the spinal cords of mice were prepared at six time points. Membrane and cytosolic fractions of cultured astrocytes were also prepared by the same procedure. A total of 20 μg protein lysates was then resolved by 10% SDS-PAGE, transferred to a PVDF membrane, and probed with rabbit monoclonal antibodies against SGK-1 (1:1,000; ab32374, Abcam, Cambridge, UK), GCR (1:1,000; sc1002, Santa Cruz Biotechnology, Santa Cruz, CA), Pannexin-1 (1:1,000; ab124131, Abcam), Actin (1:1,000; sc1616-HRP, Santa Cruz Biotechnology) or POL2 (1:1,000; sc900, Santa Cruz Biotechnology). The specificities of antibodies for their target proteins have been validated in previous studies58 (link)59 (link)60 (link). Specific antigen–antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies and a chemiluminescence reagent (Nacalai Tesque, Kyoto, Japan).