Peroxidase conjugated bovine anti mouse igg secondary antibody

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).

Total cytosolic and nuclear extracts were prepared, as previously described [26 (link)], on saphene veins. The following primary antibodies were used: anti- IL-1β (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500 #sc12742, D.B.A, Milan, Italy), anti-TNF-α (Santa Cruz Biotechnology; 1:500 #sc52746), anti-TGFβ1 (Santa Cruz Biotechnology, 1:500 #sc130348, D.B.A, Milan, Italy), anti-VEGF (Santa Cruz Biotechnology; 1:1000 #sc7269), anti- αSMA (Santa Cruz Biotechnology; 1:500 #sc53015) and anti-prolyl endopeptidase (Abcam; 1:1000,#ab58988) in 1× phosphate-buffer saline (Biogenerica srl, Catania, Italy), 5% w/v non-fat dried milk, 0.1% Tween-20 at 4 °C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1:2000) for 1 h at room temperature. Anti-β-actin (Santa Cruz Biotechnology; 1:1000 #sc47778) and anti-βTubulin (Santa Cruz Biotechnology; 1:1000 #sc5274) antibodies were used as controls. Protein expression was detected by chemiluminescence (ECL) system (Thermo, Waltham, MA, USA), visualized with the ChemiDoc XRS (Bio-Rad, USA), and analyzed by using Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA) as previously reported [27 (link)].

Cytosolic and nuclear proteins were extracted from colon tissues as previously described [28 (link)]. The behind primary antibodies were used: anti-TLR4 (SCB, 1:500, #sc293072, D.B.A, Milan, Italy); anti-Myd88 (SCB; 1:500, #sc-74532, D.B.A, Milan, Italy); anti-IKB-α (SCB, 1:500, #sc-4094, D.B.A, Milan, Italy), anti-NF-kB (SCB; 1:500 #sc8008, D.B.A, Milan, Italy), anti-Nrf2 (SCB, 1:500; #A-10:sc-365949; D.B.A, Milan, Italy); anti-HO-1 (SCB, 1:500; #sc-136960; D.B.A, Milan, Italy), in PBS with 5% w/v non-fat dried milk and 0.1% Tween-20 at 4 °C O/N. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA; 1:2000) for 1 h at room temperature. Anti-β-actin or anti-lamin A/C (D.B.A, Milan, Italy) antibodies were used as controls. Protein expression was analyzed as previously reported [34 (link)].

Western blot analysis was performed on spinal cord and brain samples with the protocol that we previously described [61 (link),62 (link),63 (link)]. Specific primary antibody: anti-P2X7R (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-NGF (1:500, Santa Cruz Biotechnology (SCB), #sc-32300), anti-c-FOS (1:500, SCB, sc-166940), anti-NLRP3 (1:1000, Cell Signaling Technology), anti-ASC (1:1000, SCB, #sc 271054), anti-Casp-1 (1:1000, Cell Signaling Technology) were mixed in 1× PBS, 5% w/v nonfat dried milk, 0.1% Tween-20, and incubated at 4 °C, overnight. After, blots were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature. To assess whether blots were loaded with equal volumes of protein lysates, we probed them with a mouse monoclonal β-actin (1:5000; SCB, #sc8432). Signals were detected with an enhanced chemiluminescence detection system reagent according to manufacturer’s instructions (Super-Signal West Pico Chemiluminescent Substrate, Pierce, Altrincham, UK) [64 (link)]. Protein expression was quantified by densitometry with BIORAD ChemiDocTM XRS+ software and standardized to β-actin levels. Images of blot signals were imported to analysis software (Image Quant TL, v2003) [65 (link)].

Lesion samples and spinal cord tissues were homogenized and Western blots were performed as already described [85 (link),86 (link)]. Specific primary antibody anti-Nox-1 (PA5-103220), anti-Nox-4 (PA5-72816), anti-MMP-9 (sc-13520), anti-iNOS (sc-7271), anti-TGF-β (sc-130348), anti-Ikb-α (sc-1643), anti-NF-kB (sc-8008), anti-COX2 (sc-376861), anti-c-FOS (sc-166940) and anti-NGF (sc-32300) was mixed in 5% w/v nonfat dried milk solution and was incubated overnight. Afterward, blots were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase conjugated goat ant-irabbit IgG (Jackson Immuno Research, Milan, Italy) for 1 h at room temperature [62 (link)]. Membranes were also blotted with the antibody against β-actin or lamin B1. Signals were detected with enhanced chemiluminescence detection system reagent (Super-SignalWest Pico Chemiluminescent Substrate) [87 (link)]. The relative expression of the protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software (Bio-Rad, Milan, Italy) and standardized to β-actin or lamin B1 levels. Images of blot signals were imported to analysis software (v2003, Image Quant TL).

A Western blot analysis was performed as already described [43 (link)]. Membranes were probed with the following primary antibodies: anti-phospho- IκB (Cell Signaling 2859), anti-MyD88 (Santa Cruz Biotechnologies, sc-74532), and anti-TLR4 (Santa Cruz Biotechnologies, sc-293072) in 1× PBS, 0.1% Tween-20, 5% w/v non-fat dried milk (PMT) at 4 °C overnight [44 (link),45 (link)]. The membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) [46 (link)]. The blots were also incubated with the primary antibody against GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA). Signals were detected with an enhanced chemiluminescence detection system reagent according to the manufacturer’s instructions (SuperSignalWest Pico Chemiluminescent Substrate, Pierce, WA, USA) [46 (link)].

Western blots were performed as described from our previous studies (Campolo et al., 2013 (link)).
Specific primary antibodies, anti-IkB-α (1:500; Santa Cruz Biotechnology), anti-NF-κB p65 (1:500; Santa Cruz Biotechnology), anti- p-JNK (1:500; Santa Cruz Biotechnology) anti-iNOS (1:500; BD Transduction), anti-p-P38 (1:1000; Cell signaling) and anti-MnSOD (1:500 Millipore) in 1x PBS, 5%(w/v) non-fat dried milk, and 0.1% Tween 20 were used at 4°C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000; Jackson Immuno Research Laboratories, West Grove, PA, USA) for 1 h at room temperature. The levels of β-actin (1:2000; Santa Cruz Biotechnology) and lamin A/C (nuclear fraction 1:500 Sigma–Aldrich, Milan, Italy) served as an internal control for protein loading. The relative expression of the protein bands of IkB-α (37 kDa), NF-κB p65 (65 kDa), iNOS (130 kDa), p-JNK (46 kDa), p-P38 (38 kDa), MnSOD (24 Kda) were detected with an enhanced chemiluminescence (ECL) system (Thermo, USA) and visualized with the Chemi Doc XRS (Bio-Rad, USA) and analyzed by using Image Lab 3.0 software (Bio-Rad, USA).