Ab179823

Renal tissues were homogenized in RIPA buffer (P0013, Beyotime, Shanghai, China) supplemented with protease inhibitors (539134, EMD Biosciences Inc., San Diego, CA). After centrifugation (12,000 rpm/min) at 4 °C for 15 min, the supernatant was added to the loading buffer. Protein samples were processed for immunoblot analysis as previously reported (Dixon et al.). The following primary antibodies were used: β-actin (AP0060; Bioworld Technology), protein kinase -like endoplasmic reticulum kinase (PERK, ab65142; Abcam), activating transcription factor 6 (ATF6, ab203119; Abcam), IRE1α (CST3294), p-IRE1α (phosphorylation sites: S724; ab124945; Abcam), JNK (CST9252), p-JNK (phosphorylation sites: Thr183/Tyr185; CST4668), CCAAT-enhancer-binding protein-homologous protein (CHOP, ab179823; Abcam), 4-hydroxynonenal (4-HNE, ab46545; Abcam), glutathione peroxidase 4 (GPX4, ab125066; Abcam). The working concentration of β-actin used was 1:5000, and that of the other antibodies was 1:1000. Secondary antibodies were obtained from Dingguo Co. Ltd. (Beijing, China) and were used at the final dilution of 1:5000. The odesay software was used for analysis (Li Cor, Lincoln, NE).

Rabbit polyclonal antibodies against GRP78 (ab188878), CHOP (ab179823), and NURR1 (ab176184); the mouse monoclonal antibody against MAP2 (ab11268); and the rabbit monoclonal antibody targeting Ki-67 (ab16667) were purchased from Abcam (United States). Rabbit polyclonal antibodies against p-eIF2α (AF3087) and ATF6 (DF6009) were purchased from Affinity (China). The Alexa Fluor 488 donkey anti-mouse IgG (H + L) (1975519) and the Alexa Fluor 594 donkey anti-rabbit IgG (H + L) (1827674) secondary antibodies were purchased from Invitrogen (United States). The immunohistochemistry kit (SP9001) was purchased from the Zhongshan Goldenbridge Biotech, China, and the morphine hydrochloride for injection was produced in the First Pharmaceutical Factory of Shenyang, China.

The hippocampus tissues were grinded with liquid nitrogen and lysed in RIPA lysis Buffer (Promega, Madison, USA), then centrifugation at 12,000 rpm for 15 min at 4°C. Total protein level was assessed via the BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Total of 30 μg proteins from each sample were run on 5% concentrated glue under 80 V for 30 min, and 12% separating gel under 120 V for 2 h. Transmembrane was performed by 100–200 mA electricity for 160 min. The membranes were blocked via 5% no-fatty milk solution in TBST (0.1% Tween20) and incubated with antibodies of anti-Grp78 (1:3000, Ab 108615, Abcam, Cambridge, MA, USA), anti-CHOP (1:3000, Ab179823), anti-Caspase-12 (1:5000, Ab62484), and anti-β-actin (1:1000, Ab13248), respectively, for overnight at 4°C. The membranes were washed three times with TBST for 10 min, and added with HRP labeled 1:5000 goat-anti-mouse second antibody for 2 h. Protein bands were shown via enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA) and visualized by a ChemiDoc MP system (Bio-Rad, Hercules, USA). The grayscale value analysis was carried out through the Quantity One image analysis system, and the relative expression of each protein was expressed by the gray value of the target protein/the gray value of the internal reference.