Cox 2

Total protein was extracted from mouse kidneys and cultured cells using RIPA Lysis Buffer (Beyotime Biotechnology, Nanjing, China). Nucleoproteins were extracted using a commercial kit (Beyotime Biotechnology). Samples (20–40 µg of protein/lane) were separated on 12% (w/v) sodium dodecyl sulfate-polyacrylamide gels; the proteins were electrotransferred to 0.22-µm pore-sized polyvinylidene fluoride membranes (Millipore Sigma, Burlington, MA, USA) and immunoblotted with primary antibodies against Klotho (Abcam; 1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA; 1:200), NF-κB p65 (Cusabio, Wuhan, China; 1:1,000), IκB-α (Abcam; 1:5,000), phosphorylated-IκB-α (Abcam; 1:1,000), COX2 (Proteintech, Rosemont, IL, USA; 1:300), caspase-3 (Proteintech; 1:1,000), Bax (Proteintech; 1:2,000), Bcl2 (Cell Signaling Technology, Danvers, MA, USA; 1:1,000), histone 3 (Cell Signaling Technology; 1:1,000), and β-actin (Servicebio; 1:2,000). They were then incubated with HRP-conjugated secondary antibodies (Proteintech; 1:10,000). The Western blots were developed using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA); band intensities were quantified with the aid of Quantity One ver. 4.6.7 software (Bio-Rad).

The protein content of the lysate was measured via the bicinchoninic acid (BCA) protein assay. Cells lysates were boiled in protein loading buffer, analyzed by SDS-PAGE, and electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were blocked with TBST (Tris-buffered saline with Tween 20) containing 5% bovine serum albumin (BSA) and incubated with primary antibodies overnight at 4°C. Immunodetection of target proteins was conducted with specific antibodies against p38 (Cell Signaling Technology), p-p38 (Cell Signaling Technology), COX-2 (Proteintech), AnxA1 (Proteintech), and GADPH (Proteintech). The blots were incubated with the appropriate secondary antibody diluted in TBST for 1 h. Immunoblots were detected via immunofluorescence.

The total, cytoplasmic, and nuclear proteins were used for western blot analysis, which were prepared as described in our previous studies [24 (link),29 (link),30 (link)]. In brief, primary antibodies for IκBα (Proteintech, Wuhan, China), NF-κB1 (p105/p50, Proteintech), RelA (p65, Proteintech), iNOS (Proteintech), IL-1β (Proteintech), IL-6 (Proteintech), TNF-α (Proteintech), COX-2 (Proteintech), MCP-1 (Abcam, Cambridge, UK), PI3K (Proteintech), Akt (Proteintech), IL-10 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), HO-1 (Proteintech), H1.2 (Proteintech), and GAPDH (Proteintech) were used in this study. The second antibody were purchased from Santa Cruz Biotechnology. In this study, the relative protein expression in group CAS-G and CAS-A was respectively set as 1.00.

Protein concentration of cells and the prefrontal cortex were determined using a BCA protein assay kit (Pierce Biotechnology, Inc.). A quantity of 20–40 μg of total proteins was loaded onto a 10–12 % gradient polyacrylamide gel, electrophoretically transferred to a polyvinylidene difluoride membrane, and probed with the following primary antibodies: TNF-α (1:1000, Santa Cruz), IL-1β (1:1000, Santa Cruz), phospho-NF-κB p65(S536) antibody (1:500, Cell Signaling Tech. MA, USA), NF-κB (1:1000, Cell Signaling), phospho-p38 antibody (1:1000, Cell Signaling), p38 antibody (1:1000, Cell Signaling), phospho-JNK antibody (1:1000, Santa Cruz Biotechnology, CA, USA), JNK antibody (1:1000, Santa Cruz Biotechnology), phospho-extracellular signal-regulated kinase (ERK)1/2 (1:2000, Cell Signaling), ERK1/2 (1:2000; Cell Signaling), OTR (1:2000, Abcam), inducible nitric oxide synthase (iNOS) (1:500, Cell Signaling), cyclooxygenase-2 (COX-2, 1:1000, Proteintech Group, Inc., CA, USA). β-actin (1:2000; Sigma-Aldrich) was used as an internal control. Secondary antibodies were horseradish peroxidase conjugated to goat/mouse anti-rabbit IgG (1:8000, Sigma-Aldrich). The membranes were developed using an enhanced chemiluminescence detection system (Pierce, Rockford, IL).

The ELISA kits of IL-6, IL-1β, TNF-α, and PGE2 were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). Dexamethasone was purchased from Shanghai Shangyao Xinyi Pharmaceutical Factory Co., Ltd. (Shanghai, China). Carrageenan and aspirin were purchased from Macklin Reagent Co., Ltd. (Shanghai, China). Xylene was obtained from Hangzhou Shuanglin Chemical Co., Ltd. (Hangzhou, China). Ammonia was purchased from Hangzhou Longshan Fine Chemical Co., Ltd. (Hangzhou, China). Citrate buffer solution and DAB were obtained from Beyotime Biotechnology Reagent Co., Ltd. (Zhejiang, China). BSA and anti-rabbit IgG were obtained from Boster Biological Technology Co., Ltd. (Zhejiang, China). COX-2, NF-κB p65, and GAPDH antibodies were purchased from Proteintech Biotechnology Co., Ltd. (Zhejiang, China).

A phenolmethylsulfonyl–fluoride-containing lysis buffer containing radioimmunoassay precipitation was used to extract the total protein from glioma cells (Servicebio, Wuhan, China). In order to determine the protein concentrations in the samples, bicinchoninic acid (BCA) protein assays were performed (Servicebio, Wuhan, China). Separating proteins with sodium dodecyl sulfate polyacrylamide gels (Thermo Fisher Scientific, Waltham, MA, USA) led to their transfer to polyvinylidene fluoride membranes. Using skimmed milk powder to block the membranes, primary antibodies containing SLC7A11 (Cat No. 26864-1-AP), COX2 (Cat No. 66351-1-Ig), GPX4 (Cat No. 67763-1-Ig), JUN (Cat No. 66313-1-Ig), and β-actin (Cat No. 81115-1-RR) were purchased from proteintech; SLC40A1 (Cat No. ab239583) was purchased from abcam, in a dilution of 1:1000, was added for 2 h at room temperature and kept overnight at 4 °C. In total, 2 h of incubation was conducted at room temperature with a secondary antibody (in a dilution of 1:2000) after washing three times. A high-sensitivity ECL exposure solution was added and developed using an imager. β-actin was used as the loading control to calculate the relative protein expression.