Phospho ikkβ

The cells were harvested after stimulation and washed three times by ice-cold PBS (MDL biotech, Beijing, China) on ice, and the cells were lysed with RIPA buffer (MDL biotech, Beijing, China) with proteasome inhibitor cocktail (Roche, Indianapolis, IN), followed by sonication. After centrifugation for 15 min at 13,000 g, supernatants were collected and the protein concentration of lysates was measured using Bio-Rad quantification assay (Bio-Rad Laboratories, Hercules, CA). Twenty micrograms of protein sample was incubated with loading buffer (Beyotime, Beijing, China) at 95°C for 10 minutes. Proteins were separated using 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, USA). The membrane was then blocked with 2.5% nonfat dry milk for 1 h. The antibodies specific for p65, phospho-p65, IκBα, phospho-IκBα, IKKβ, phospho-IKKβ (Cell Signaling Technology Inc., Beverly, USA), and β-Actin (Santa Cruz Biotechnology, Santa Cruz, USA) were added and incubated overnight at 4°C. After incubation with the corresponding horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, USA), the target protein was visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Bremen, Germany).

Human thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). Tunicamycin was purchased from Sigma. Polyclonal antibodies to ICAM-1, VCAM-1, RelA/p65, β-actin, IκBα, and Lamin B were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to phospho-eIF2α, eIF2α, BiP/GRP78, ATF-4, phospho-AKT, phospho-IKKβ, IKKβ, phospho-(Ser⁵³⁶)-RelA/p65, were obtained from Cell Signaling (Beverly, MA). Tata Binding Protein (TBP) antibody was purchased from Abcam. RelA/p65 transcription factor assay kit was purchased from Cayman Chemical (Ann Arbor, MI) and plasmid maxi kit was from QIAGEN Inc. (Valencia, CA). Protein assay kit and nitrocellulose membrane were from Bio-Rad. Alexa Fluor 488-phalloidin was purchased from Invitrogen. Expression vector encoding Wild type BiP and dominant negative BiP were from Addgene. All other materials were from VWR Scientific Products Corporation (Gaithersburg, MD) and Fisher Scientific (Pittsburgh, PA). SubAB and its non-toxic derivative SubA_A272B were purified as previously described [37] (link).

Rhein were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of the compound rhein was certified to be 98% by high-performance liquid chromatography (HPLC). Lipopolysaccharide (LPS) from Escherichia coli 055:B5 was obtained from Sigma-Aldrich Chemical Co. (USA). TNF-α, IL-1β, monocyte chemoattractant protein (MCP)-1 and IL-8 ELISA Kits were purchased from Yantai Science & Biotechnology (Shandong, China). Antibodies against phospho-IκBα, phospho-NF-κB p65 and phospho-IKKβ were obtained from Cell Signaling Technology (Beverly, MA, USA). Blood Urea Nitrogen (BUN) and Serum Creatinine Determination (SCr) assay kit reagents were supplied by were purchased from the Institute of Jiancheng Bioengineering (Nanjing, China). DMEM medium and fetal bovine serum (FBS) were products of Gibco Corporation (USA). Zymosan A, nitroblue tetrazolium (NBT) and the other reagents were all purchased from Sigma-Aldrich Chemical Co. (USA). All other reagents were of analytical grade.

Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum and the antibiotic-antimycotic solution were purchased from Gibco (Auckland, New Zealand). The ELISA kits for TNF-α, IL-1β and IL-6 were obtained from Neobioscience (Beijing, China). Primary antibodies against extracellular signal-regulated kinase (ERK), phospho-ERK, p38, phospho-p38, c-Jun N-terminal kinase (JNK), phospho-JNK, IκB-α, phospho-IκB-α, p65, phospho-p65, IKKβ and phospho-IKKβ were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-conjugated secondary antibodies were also obtained from Cell Signaling Technology.

Formamide was provided by Sigma-Aldrich (Guangzhou, Guangdong, China). Cell lysis buffer was ordered from Beyotime Biotechnology (Shanghai, China). Antibodies for β-actin, cyclooxygenase 2 (COX-2), 5-Lipoxygenase (LOX), Erk, Phospho-Erk, JNK, Phospho-JNK, p38, Phospho-p38, IKKβ, Phospho-IKKβ, IκBα, Phospho-IκBα (Ser32/36), NF-κB p65, and phospho-NF-κB p65 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Relative peroxidase-conjugated secondary antibody, Bicinchoninic acid (BCA) assay, Evans blue (EB) dye and Pierce™ ECL Western Blotting Substrate were purchased from Thermo Scientific (Waltham, MA, USA). ELISA for IL-6, TNF-α, PGE₂, LT-B₄ and LT-D₄ were purchased from Cusabio (Wuhan, Hubei, China).

Cells were harvested and washed with ice cold PBS for three times. Frozen kidney tissues were thawed and then homogenized mechanically to extract protein. Cytoplasmic and nuclear extracts were gained by utilizing a cytoplasmic/ nuclear isolation kit (Beyotime Institute of Biotechnology, Beijing, China). Proteins were segregated by electro-blotted and SDS-PAGE into a membrane of nitrocellulose. The blots were probed with antibodies against IκBα, IKKβ, phospho-IκBα, phospho-IKKβ and p65 (all from Cell SignalingTechnology, Beverly, MA, America) at 4°C overnight and later incubated with horseradish peroxidase-conjugated anti-IgG (Boster Biotechnology, Wuhan, Hubei province, China) as secondary antibody solution at room temperature for 60 min. The expression of every protein could be detected by the detection system of ECL (ChemiDoc™XRS, Bio-Rad). The respective bands were quantitated and images were collected utilizing Quantity One Software (Bio-Rad). The fold increase over control could be used to express the results.