Ab227828

Total protein extracts were isolated using RIPA lysis buffer (Sigma, USA), and were quantified with bicinchoninic acid (BCA) kit (Pierce, USA). Afterwards, extracted proteins were separated via SDS/PAGE, and transferred onto PVDF membranes. The membranes were then blocked by TBST supplemented with 5% skimmed milk. Antibodies listed below were utilized for incubating the membranes, comprising ADAR1 (1/1000; ab168809; Abcam), β-actin (1/5000; ab179467; Abcam), Keap1 (1/2000; ab227828; Abcam), and Nrf2 (1/500; ab137550; Abcam), and HRP anti-rabbit IgG antibody (1/2000; ab288151; Abcam).

HaCaT and PIG1 cells were lysed in RIPA lysis buffer on ice. Equal amounts of protein from each sample were separated using 10% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). In addition, 5% bovine serum albumin (BSA) (Yeasen, Shanghai, China) was used to block nonspecific binding for 1 h. The membranes were probed with anti-Keap1 (ab227828), anti-Nrf2 (ab62352), anti-HO-1 (ab13248), and anti-β-actin (ab8227) antibodies overnight at 4°C; these primary antibodies were obtained from Abcam (Cambridge, MA, USA). Membranes were then incubated with secondary antibodies, including goat anti-mouse (115-035-003; Jackson ImmunoResearch, PA, USA) and goat anti-rabbit (111-035-003, Jackson ImmunoResearch, PA, USA) antibodies, at room temperature for 1 h. Thereafter, the protein bands were examined using an ECL western blot detection system (Merck Millipore, Burlington, MA, USA). The antibodies used are listed in Supplementary Table 2.

Neonatal rat hippocampal tissues were extracted and total protein was obtained using RIPA buffer containing PMSF. The protein concentration was determined using the BCA assay kit (Beyotime, China) on postnatal day 21. Subsequently, the protein loading buffer was added and the samples were denatured at 95°C for 10 min. After that, the proteins were isolated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to an activated PVDF membrane. Following blocking, the membrane was incubated overnight at 4°C with the appropriate concentration of primary antibody. Subsequently, the fluorescently labeled secondary antibody (IRDye700 and IRDye800, goat anti-rabbit) was incubated for 1 h at 37°C. Color rendering was performed using a chemiluminometer (Licor, America). The primary antibodies used were as follows: anti-NeuN (ab177487, abcam), anti-GPX4 (ab125066, abcam), anti-FTH1 (ybs-5907R, YOYOBIO), anti-PTGS2 (ab226869, abcam), anti-ACSL4 (PA5-27137, Invitrogen), anti-Nrf2 (PA5-27882, Invitrogen), and anti-Keap-1(ab227828, abcam).

Total protein extracts were prepared by homogenization of mouse ventricles in Urea lysis buffer. Samples were separated on 4-12% SDS-PAGE gels (Life Technologies) and incubated with primary antibodies, including STK3 (1:1500, #ab52641, Abcam), KEAP1 (1:1500, #ab227828, Abcam), Bax (1:2000, #ab32503, Abcam), Bcl2 (1:1500, #ab182858, Abcam), and Nrf2 (1:1500, #ab92946, Abcam). Immunoreactive protein bands were visualized using ECL reagent. The representative image for each group was selected to show the average or median level of the group based upon the mean value 34 (link).

The protocol was modified based on publications [25 (link),26 (link)]. Proteins of cells were lysed and collected with RIPA lysis solution. Protein concentration was determined by the BCA method. The protein samples of each group were loaded in equal quantities and transferred to the polyvinylidene fluoride membrane (BD Biosciences, USA) after electrophoresis. The membrane was blocked with QuickBlock™ blocking buffer (Beyotime) at room temperature and incubated overnight with a specific primary antibody for Keap1, Nrf2, HO-1, and β-actin (#ab227828, #ab137550, # ab13243, and #ab115777, Abcam, UK) at 4°C. TBST was used for a full washing, and the corresponding secondary antibody was added and incubated at room temperature for 1 h. After washing, ECL luminescent solution (Millipore, USA) was dropped to visualize protein bands. β-actin served as the internal reference.

The IHC was carried out according to standard protocol. In shortly, all paraffin specimens were sliced to a thickness of 4 µm, then deparaffinized and hydrated and heated in Tris/EDTA buffer pH 9.0 for antigen retrieval. The Keap1 antibody (1:500, ab227828, Abcam) was incubated overnight at 4 °C and the specimens were visualized with a light microscope (Olympus, Tokyo, Japan). Two pathologists completed the readings independently. The average number of positive cells in each field was analyzed as the percentage of positive cells in the section and scored using the Fromowitz criteria (Fromowitz et al., 1987 (link)): 0–5% was scored as 0, 6–25% was scored as 1, 25–50% was scored as 2, 51–75% was scored as 3, and >75% was scored as 4. The intensity of staining was scored according to the staining characteristics of most positive cells: 0 score for no staining, 1 score for light yellow, 2 score for brown, and 3 score for dark brown. The final score was derived by summing the percentage of stained cells and the corresponding intensity, with 0–1 being classified as negative (−), 2–3 as weakly positive (+), 4–5 as moderately positive (++), and 6–7 as strongly positive (+++). Where (−) and (+) recorded as low expression, (++) and (+++) recorded as high expression groups.