Ab92946

The homogenate from the liver tissue was collected, and the lysate was used to lyse the hepatocytes for 30 min on ice. The BCA method was used to determine the protein concentration. The sample was added to 5× loading buffer by volume and heated in a water bath at 100 °C for 5 min. The hepatocytes were sonicated by SJIALAB for 2 min (10% ultrasound intensity) and centrifuged for 10 min to obtain the supernatant. The extracted protein was subjected to polyacrylamide gel electrophoresis (80–120 V), transferred to a PVDF membrane, sealed and incubated overnight at 4 °C with the addition of primary antibody of nNOS (3G6B10, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), eNOS (9D10, Invitrogen), iNOS (PA1036, Invitrogen), NF-κB (PA1186, Invitrogen), IκB-α (397700, Invitrogen), Mn-SOD (PA530604, Invitrogen), Cu/Zn-SOD (PA5270240, Invitrogen), CAT (PA259183, Invitrogen), HO-1 (ab68477, Abcam, Cambridge, MA, USA), Nrf2 (ab92946, Abcam), γ-GCS (sc-390811, Santa Cruz Biotechnology, Dallas, TX, USA), NQO1 (ab28947, Abcam) and β-action (MA5157739, Invitrogen). The protein was incubated overnight at 37 °C with the addition of a second antibody (A21241) for 2 h, and color detection and chemiluminescence imaging analysis system (Tanon 6200, ShanghaiTanon Technology Co., Ltd., Shanghai, China) were used to collect images [56 (link)].

Western blot analyses were performed as previously described31 (link). Briefly, cerebral cortex samples were collected, homogenized, and extracted in RIPA lysis buffer containing a 1% (v/v) protease and phosphatase inhibitor cocktail. The protein samples were separated by SDS–PAGE and transferred to PVDF. After blocking with 5% (w/v) nonfat milk, the membranes were incubated with the following primary antibodies overnight at 4°C: rabbit anti-β-actin (1:1,000, rabbit polyclonal, Abcam, ab8227), rabbit ZO-1 (1:1,000, rabbit polyclonal, Abcam, ab 96587), phospho-p38 (CST, #4551, 1:2,000), and rabbit anti-Nrf2 (1:1,000, rabbit polyclonal, Abcam, ab92946). Afterwards, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (1:5,000) for 1 h at room temperature and then scanned with a Bio-Rad (California, United States of America) gel imaging system.

Total protein and nucleoprotein (for Nrf2 and β-TrCP) in the ipsilateral peri-infarct hemisphere were extracted, and the protein concentration was determined by bicinchoninic acid protein (BCA) assay. The protein samples were then denatured by boiling for 10 min. Then, 20 μg of each protein sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 80 V for 100 min. After transfer to polyvinylidene fluoride (PVDF) membrane, the separated protein bands were incubated with individual primary antibodies (caspase-3 1:500, ab197202, Abcam; AKT, 1:1000, Cat# 9272, CST; p-AKT, 1:1000, Cat#9271, CST; GSK-3β, 1:1000, Cat# 9315, CST; p-GSK-3β, 1:1000, Cat# 9336, CST; β-TrCP, 1:1000, Cat# 4394, CST; Nrf2 1:1000, ab92946, Abcam; PCNA, 1:1000, ab92552, Abcam; HO-1, 1:2000, ab52947, Abcam; NQO1, 1:10000, ab80588, Abcam) at 4 °C overnight, followed by incubation with corresponding secondary antibodies at room temperature for 2 h. The developed bands were observed under a gel imaging system.