Nf κb p65 antibody

The following reagents were purchased: docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA, Cayman Chemical); Poly(dA:dT), adenosine triphosphate (ATP), and nigericin (Sigma-Aldrich); Lipopolysaccharide (LPS, ENZO Life Sciences); purified flagellin (InvivoGen); neutralizing antibody to IL-1β (R&D Systems); caspase-1 antibody (Santa Cruz Biotechnology); NLRP3 antibody (ENZO Life Sciences); ASC antibody (Santa Cruz Biotechnology); and the appropriate secondary antibodies (Jackson ImmunoResearch Laboratory). For the ImageStream analysis a primary rabbit polyclonal NF-κB/p65 antibody (SantaCruz Biotechnology) was used with an Alexa647 conjugated donkey anti rabbit IgG antibody (Jackson ImmunoResearch Laboratories). DNA was stained using DAPI (20 µM).

Cells were plated onto poly-l-lysine coated glass slides and fixed in 4 % (v/v) methanol free formaldehyde solution (pH 7.4) at 4 °C for 25 min. The cells were permeabilized in 0.2 % (w/v) Triton X-100, blocked in 5 % (w/v) bovine serum albumin (BSA) in humidified chamber, followed by immunostaining with NF-κB p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and Texas Red-conjugated secondary antibody. The cells were mounted with mounting medium with coverslips with a mounting medium containing DAPI (Vector Laboratories, Inc, Burlingame, CA) and visualized under an FlouviewFV10i confocal microscope (Olympus, Tokyo, Japan).

Nuclear proteins were isolated from iris and ciliary body by using ReadyPrepTM Protein Extraction Kit (Cytoplasmic/Nuclear, Bio-Rad, Hercules, CA, USA). Protein concentration was adjusted equally with protein assay kit (Bio-RAD, Hercules, CA, USA), then re-suspended in 5x sample loading buffer, heated for 5 min at 95°C and separated on 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto a nitrocellulose membrane (AmershamTM HybondTM-ECL, GE Healthcare, UK), blocked with 5% Bovine albumin BSA (A9418, Sigma-Aldrich, USA), and incubated with NF-κBp65 antibody (1∶300, sc-372, Santa Cruz Biotechnology, INC.) and Lamin B antibody (1∶500, sc-6216, Santa Cruz Biotechnology, INC.) at 4°C overnight. After washing with TBS-0.05% tween-20 (TBST), HRP-coupled secondary antibodies (1∶1000, Santa Cruz Biotechnology, INC.) were applied to the membrane for 1 hour at room temperature, followed by three washes with TBST. The immunoreactive bands were visualized with enhanced chemiluminescence reagents (GE Healthcare, UK) and images were captured by the Universal Hood II image system (Bio-Rad Laboratories, Segrate, Italy). Band intensities of NF-κBp65 were normalized with those of internal control (Lamin B) using NIH Image J software (version 1.47).

Renal tissues were lysed with RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in PBS, pH 7.4) containing 0.1 mM vanadyl sulfate and protease inhibitors (0.5 mg/ml aprotinin, 0.5 mg/ml trans-epoxy succinyl-L-leucylamido-(4-guanidino)butane (E-64), 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 ng/ml leupeptin). Tissue lysates (300 μg) were precleared with protein A/G-agarose and then incubated with either agarose-conjugated VDR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or NF-κB p65 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 °C overnight. The precipitates were washed with cold RIPA buffer before immunoblots using either NF-κB p65 or VDR antibody.

7R-maresin 1 was bought from Cayman Chemical (Ann Arbor, MI, USA). Alanine transaminase (ALT), aspartate transaminase (AST), creatinine (Cre), and blood urea nitrogen (BUN) detection kits were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Mouse tumor necrosis factors-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Dakewe Bioengineering Co., Ltd. (Shenzhen, China). NF-κb p65 antibody and β-actin were obtained from Santa Cruz Biotechnology. Lamin B1 antibody was purchased from Epitomics (Burlingame, CA).