Anti nf kb

Western blots were performed as described from our previous studies [60 (link)]. Specific primary antibody: anti-NRF2 (1:600, Santa Cruz Biotechnology) or anti-iNOS (1:700; Santa Cruz Biotechnology) or anti-IkB-α (1:700; Santa Cruz Biotechnology) or anti-NF-kB (1:700; Santa Cruz Biotechnology) was mixed in 1 × PBS, 5% w/v nonfat dried milk, and 0.1% Tween 20, and were incubated at 4 °C, overnight. Afterwards, blots were incubated with peroxidase-conjugated bovine antimouse IgG secondary antibody or peroxidase-conjugated goat antirabbit IgG (1:2000, Jackson Immuno Research) for 1 h at room temperature. To verify the equal amounts of protein, membranes were also incubated with the antibody against laminin (1:1000; Santa Cruz Biotechnology) and GADPH (1:1000; Santa Cruz Biotechnology). Signals were detected with enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate, Pierce). The relative expression of the protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software and standardized to β-actin levels. Images of blot signals were imported to analysis software (Image Quant TL, v2003).

The intervertebral discs were collected during surgery from patients during ACF treatment. Samples were homogenized and lysed. Extracts were resolved on SDS-polyacrylamide gels followed by transfer to nitrocellulose membranes. Proteins were resolved by electrophoresis on 8–12% sodium dodecyl sulfate–polyacrylamide gels and transferred by electroblotting to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk and incubated overnight at 4°C with the anti-HIF-1α (Novus Biological, 1;1000), anti-vascular endothelial growth factor (anti-VEGF) (Santa Cruz, 1∶1000), anti-VEGF receptor (anti-VEGFR) (Santa Cruz, 1∶1000), anti-NF-kB (Santa Cruz, 1∶1000), anti-interleukin 1 (anti-IL1) (Santa Cruz, 1∶1000), anti-interleukin6 (anti-IL6) (Santa Cruz, 1∶1000), anti-Osteopontin (OPN) (Santa Cruz, 1∶1000), anti-Osteoprotegerin (OPG) and anti-GAPDH (Santa Cruz, 1∶2000), antibodies. Immunolabeling was detected using the enhanced chemiluminescence Reagent (Amersham Biosciences).

Whole cell lysates were prepared using RIPA buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) with added protease inhibitors (Roche, Indianapolis, IN). Nuclear protein extracts were prepared using the Nuclear/Cytosol Fractionation Kit protocol (Biovision, Mountain View, CA). Thirty micrograms of protein were subjected to electrophoresis for 45 min at 200 V on a SDS polyacrylamide gel and transferred to a PVDF filter. The filters were blocked with 3% BSA for 1 h and then incubated overnight with anti-MAOA polyclonal antibody, anti- NFkB (Santa Cruz Biotechnology, Santa Cruz, CA), anti-HIF-1α (BD Biosciences, Franklin Lakes, NJ), or with anti-β-actin goat antibody (Promega Corp, Madison, WI). The filters were then incubated with species appropriate horseradish peroxidase labeled secondary antibodies (Thermo Scientific, Rockford, IL). Immuno blots were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL).

Western blotting (WB) was performed to analyse protein expression of COMP-2 and NF-kB as key biomarkers of inflammation. Intracellular proteins were obtained using Radio-Immunoprecipitation Assay (RIPA buffer) (1×) (Cell Signaling Technology, Danvers, MA, USA) and Bradford method was used to quantify their concentrations. 10% SDS-PAGE was performed to electrophoretically resolve 2 μg of proteins and transfer them to nitrocellulose membrane (GE, Amersham, UK). This latter was blocked with 5% nonfat milk in Tris-buffered saline and 0.05% Tween-20 (TBST) and it was incubated with. primary antibodies (1:500 dilution) anti- COMP-2 and anti-NF-kB (Santacruz Biotechnology, Dallas, TX, USA). Immunoreactive bands, after TBST washing, were revealed using horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution) (Santacruz Biotechnology, USA). ECL (Millipore, USA) was used to obtain the specific signals. Normalization was performed with respect to Actin (1:500 dilution) housekeeping protein (Santacruz Biotechnology, USA).

Proteins were extracted from HCN cells and the protein concentrations were assessed using a BCA assay kit (TaKaRa BIO INC, Japan). The protein samples were resolved in a 10-12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene fluoride (PVDF) membrane, and blocked with 5% nonfat milk in Trisbuffered saline-Tween (TBST) 20 for 2 h at room temperature. Membranes were then incubated with primary antibody overnight. The antibodies were shown as follows: anti-Caspase-3, anti-Bcl2, anti-IL1, anti-IL6 and anti-NF-kb (1:1000; Santa Cruz Biotechnology,USA). An anti-GAPDH antibody was used as a loading control. Membranes were washed and incubated for 2 h in the presence of appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. The positive reaction was visualized by using 3, 3’-diaminobenzitine (DAB) solution (Sigma, St. Louis, MO) with a chemiluminescent Immobilon Western blotting detection system.

Spinal cord and lumbar disc 5–6 tissues were homogenized, and Western blots were performed as already described [79 (link),80 (link),81 (link)]. The samples were stored at −80 °C for further analysis.
Specific primary antibodies against Aggrecan (sc-166951, Santa Cruz Biotechnology), NGF (MA5-32067, Thermo Fisher, Milan, Italy), trkA (JJ084-04, Thermo Fisher), WNT3a (sc-80457, Santa Cruz Biotechnology), anti-FZ8 (Bioworld Technology, St. Louis Park, MN, USA), anti–β-catenin (610153, BD Biosciences), anti-active β-catenin (05-665, Millipore, Milan, Italy), anti-NFkB (Santa Cruz Biotechnology, sc-8008), anti-NOS2 (Santa Cruz Biotechnology, sc-7271), or anti-COX-2 (Santa Cruz Biotechnology, sc-376861) were mixed in a 5% w/v nonfat dried milk solution and incubated with the membranes at 4 °C overnight. Afterwards, the blots were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibodies or peroxidase-conjugated goat anti-rabbit IgG (Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature [82 (link)]. The membranes were also incubated with antibodies against β-actin or lamin A/C (Santa Cruz Biotechnology) to verify that equal amounts of protein were loaded [83 (link)]. Images of the blot signals were imported into an analysis software (v2003, Image Quant TL, Milan, Italy) [84 (link)].

Western blots were performed as already described [74 (link),75 (link)]. Specific primary antibody: each antibody, anti-Nrf2 (Santa Cruz Biotechnology, sc-365949), anti-HO-1 (Santa Cruz Biotechnology, sc-390991), anti-NQO-1 (Santa Cruz Biotechnology, sc-32793), anti-IkB-α (Santa Cruz Biotechnology, sc-1643), anti-Nf-kb (Santa Cruz Biotechnology, sc-8008), anti-COX-2 (Santa Cruz Biotechnology, sc-376861), anti-Bcl-2 (Santa Cruz Biotechnology, sc-7382), anti-Bax (Santa Cruz Biotechnology, sc-7480), anti-MMP2 (Santa Cruz Biotechnology, sc-13594), anti-p-AKT (Abcam), anti-AKT (Abcam), was mixed in a 5% w/v of non-fat dried milk solution and incubated at 4 °C, overnight. Afterwards, blots were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (Jackson Immuno Research) for 1 h at room temperature. To verify the equal amounts of protein, membranes were also incubated with an antibody against β-actin (Santa Cruz Biotechnology). Signals were detected with an enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate, Pierce). The relative expression of the protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software and standardized to β-actin levels [76 (link)]. Images of blot signals were imported to analysis software (Image Quant TL, v2003).