Nf κb

BV-2 cells were plated in a 100-mm culture dish at a density of 2 × 10⁶ cells, activated with 10 ng/mL LPS, and treated with either WV or BV at the designated concentrations for 24 h. The cells were collected and fractionated using NE-PER^® Nuclear and Cytoplasmic Extraction Reagent Kits (Thermo Fisher Scientific, Rockford, IL, USA). The cytoplasmic and nuclear fractions of the proteins were blotted and analyzed for the expression levels of various proteins, as previously described [39 (link),40 (link)]. The primary antibodies used were immunoglobulins against iNOS (Enzo Life Sciences, Farmingdale, NY, USA), COX-2 (Cell Signaling Technology, Danvers, MA, USA), NF-κB (BioWorld Technology, St. Louis, MN, USA), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), and Lamin B (Santa Cruz Biotechnology, Dallas, TX, USA). After allowing the appropriate secondary antibody (horseradish peroxidase conjugate) to interact with the primary antibody, protein bands were visualized using the SuperSignal™ West Pico Chemiluminescent Substrate (Pierce, Cheshire, UK) and LAS4000 Mini (GE Healthcare Life Sciences, Little Chalfont, UK). The digitalized blot images were then densitometrically analyzed using Image-Studio Lite version 5.2 (LI-COR Biotechnology, Lincoln, NE, USA).

Cytoplasmic and nuclear fractions of cultured cells and mouse tissues were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Rockford, IL, USA). The proteins in each fraction were blotted and their relative expression levels were determined as previously described [34 (link),35 (link)]. Briefly, the primary antibodies used in this study were immunoglobulins against COX-2 (Cell Signaling Technology, Danvers, MA, USA), NF-κB (Bioworld Technology, St. Luis. MN, USA), iNOS (Enzo Life Sciences, Farmingdale, NY, USA), HO-1 (Abcam, Cambridge, UK), Nrf2 (Abcam, Cambridge, UK), β-actin, and Lamin B1 (Santa Cruz Biotechnology, Dallas, TX, USA). They were used at a dilution ratio of 1:1000 in 1% bovine serum albumin (Bio Basic Inc., Markham, ON, Canada) in tris-buffered saline (TBS). After allowing the appropriate secondary antibody (horse radish peroxidase–conjugated) at 1:2000 in TBS to interact with the primary antibody, protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Cheshire, United Kingdom) and LAS4000 Mini (GE Healhcare Life Sciences, Little Chalfont, UK). The digitalized blot images were then densitometrically analyzed using Image-Studio Lite version 5.2 (LI-COR Biotechnology, Lincoln, NE, USA).

Nuclear proteins and total cell lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene fluoride membrane (PVDF; Merck Millipore, Billerica, MA, USA) at 100 V (1 h) in buffer containing 0.3% Tris, 1.4% glycine, and 20% methanol using a wet-blotting apparatus (Mini-PROTEAN Tetra cell; Bio-Rad, Hercules, CA, USA). The PVDF membrane containing the transferred proteins was blocked with 5% BSA in PBS for one hour at room temperature. Primary monoclonal antibodies against human TBK1 (Abcam, Inc.) and NF-κB (Bioworld Technology, Inc.) diluted in PBS (1 : 1000) were applied to the PVDF membrane and incubated overnight at 4°C. Secondary antibodies diluted in PBS (1 : 2000) were subsequently applied to the PVDF membrane and incubated for 1 h at room temperature. The PVDF membrane was washed four times (10 min each) with Tris-buffered saline (TBS; 50 mM Tris HCl pH 7.5, 150 mM NaCl) containing 0.1% Tween 20. The binding of specific antibodies was visualized using an enhanced chemiluminescence western blotting detection kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Densitometric quantification of the immunoblot was carried out using ImageJ software. The value of each band was normalized to β-actin or lamin.