P nf κb p65

Cells were lysed in ice-cold radioimmunoprecipitation(RIPA) lysis buffer (Catalog No. 89901, Thermo Scientific, Rockford, USA) containing protease inhibitor cocktail (11873580001, Roche Diagnostic, Germany) and phosphatase inhibitor cocktail (P5726, Sigma-Aldrich). Protein concentrations were determined by BCA protein assay kit (Catalog No. 23225, Thermo Scientific, Rockford, USA) as described by manufacturer instructions. Equal amounts of proteins were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (PVDF). After blocking, membranes were incubated with primary antibodies such as p-p38 (9211S, Cell Signaling), p38 (9212S, Cell Signaling), p-ERK1/2 (9101S, Cell Signaling), ERK1/2 (9102S, Cell Signaling), p-NF-κBp65 (SC-136548, Santa Cruz), NF-κBp65 (SC-109, Santa Cruz) and GAPDH (SC-32233, Santa Cruz) at 1:1,000 dilutions for overnight at 4°C. Membranes were then washed and incubated with horseradish-peroxidase conjugated secondary antibodies (1:5,000 dilutions) for 2 h at room temperature. The blots were then detected with western blot detection kit (WesternBrightTMECL, USA).

After 12 weeks of treatment, the kidneys from each of the mice were collected and prepared using a protein extraction kit (KetGEN Biotech Inc., Nanjing, China) according to the manufacturer’s protocol. Equal quantities of protein were separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumen (Amresco, Inc. USA) and incubated with the primary antibodies: AGEs (Abcam, UK), RAGE (Abcam, UK), PI3K p85 (cell signaling technology, USA), p-PI3K p85 (cell signaling technology, USA), Akt (pan) (cell signaling technology, USA), p-Akt (Ser473) (cell signaling technology, USA), IκBα (cell signaling technology, USA), NF-κB p65 (cell signaling technology, USA), p-NF-κB p65 (Santa Cruz Biotechnology, Glostrup, Denmark) and GAPDH (Beijing Zhong Shan Golden Bridge Biotechnology Co., Ltd., China), with a dilution of 1:1000, overnight at 4 °C. Following incubation with a horseradish peroxidase-labeled secondary antibody (Beijing Zhong Shan Golden Bridge Biotechnology Co., Ltd.,) at room temperature for 1 h, the membranes were developed with enhanced chemiluminescence (Thermo Scientific, USA) and visualized using a digital imaging system (BIO-RAD Laboratories, Inc., USA).

Cells were lysed with buffer containing 1% NP-40 and proteinase inhibitor cocktail (Thermo Scientific, Rockford, IL). Protein concentrations were determined by Bradford assay (Bio-Rad) and equalized before loading. Cellular protein from each sample was applied to 8% to 12% SDS-PAGE gels and probed with specific antibodies including EEA1, Rab7, RIP1 (Cell Signaling Technology, Danvers, MA), AnxA2, p-IκBα, p-IKKα, p-ERK, p-p38, p-JNK, p-NFκB p65, p-NFκB p50, TNFα, IL-6, IL-1β, and β-actin (Santa Cruz Biotechnology). Blots were developed with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and chemiluminescent substrate on Fuji X-ray films52 (link).

Cells were harvested after 5 days of incubation at 37 °C in humidified incubator and were lysed in ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Catalog No. 89901, Thermo Scientific, Rockford, USA) containing protease and phosphatase inhibitor cocktails (Roche Diagnostic, Germany). Protein concentrations were determined by BCA protein assay Kit (Catalog No. 23225, Thermo Scientific, Rockford, USA) as described by manufacturer instructions. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (PVDF). After blocking, membranes were incubated with primary antibodies such as p-p38 (9211S, Cell Signaling), p38 (9212S, Cell Signaling), P-JNK (9251S, Cell Signaling), JNK (9252S, Cell Signaling), P-JAK1 (3331S, Cell Signaling), JAK1 (3332S, Cell Signaling), P-JAK3 (5031S, Cell Signaling), JAK3 (8863S, Cell Signaling) P-STAT6 (9361S, Cell Signaling), STAT6 (9362S, Cell Signaling), p-NF-κBp65 (SC-136548, Santa Cruz), NF-κBp65 (SC-109, Santa Cruz) and GAPDH (SC-32233, Santa Cruz) at 1:1000 dilutions for overnight at 4 °C. Membranes were then washed and incubated with horseradish-peroxidase conjugated secondary antibodies (1:5000 dilutions) for 2 h at room temperature. The blots were then detected with western blot detection kit (WesternBrightTMECL, USA).

The experiment was terminated by performing Western blot analysis of collected lung tissue samples, as described in the literature [25 (link)]. The samples were homogenized, lysed, and centrifuged using differential centrifugation for obtaining different protein components. Protein concentrations were measured by a BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Membranes were incubated with the primary antibodies against His-probe, cleaved caspase-3, Bcl-2, Bax, NF-κBp65, phosphorylated NF-κBp65 (p-NF-κBp65), and TLR4 (Santa Cruz Biotechnology, CA, USA) overnight at 4°C. Then, the membranes were washed in phosphate-buffered saline (0.05%). Tween 20 and proteins bands were visualized with an enhanced chemiluminescence kit (Amersham, Piscataway, NJ, USA). All band densities were quantified by densitometry using the Quantity One software (BioRad, Hercules, CA, USA).

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Escherichia coli lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Interleukin-1 beta (IL-1β), IL-6, IL-10, and tumor necrosis factor alpha (TNF-α) Elisa kits were obtained from eBioscience (Science Center Drive San Diego, CA, USA). COX-2 Elisa kit was purchased from Axygen (Central Avenue Union City, CA, USA). Antibodies against IκBα, p-IκBα, NF-κB p65, p-NF-κB p65, p38α, p-p38α, JNK1/2, p-JNK1/2, ERK1/2, p-ERK1/2, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and secondary antibodies were obtained from Sangon Biotech (Shanghai, China). Other chemicals and reagents were all analytically pure and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

The protease inhibitor cocktail, PMSF, and EDTA were obtained from Sigma Aldrich (Seoul, Korea). LPS was purchased from Sigma Chemical Company. The Cell Viability, Proliferation & Cytotoxicity Assay Kit was purchased from DoGenBio Co., Ltd. (Seoul, Korea). The bovine serum albumin standard, protein assay reagent, and PVDF were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Griess reagent was obtained from Promega (Madison WI, USA). iNOS, COX-2, β-actin, p-IκBα, p-NF-κB p65, IL-1β, and TNF-α antibodies and Luminol Reagent were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).