Ecl advance

Cell lysates were prepared in cell lysis buffer (Cell Signaling, Danvers, MA, USA). Protein concentration was determined by the micro bicinchoninic acid method (Pierce, Rockford, IL, USA).
Equal amounts of proteins (25–40 μg) were subjected to SDS-PAGE and transferred onto nitrocellulose membrane. Then membranes were saturated with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 and incubated overnight with primary antibody at 4°C and subsequently with horseradish peroxidase-conjugated secondary antibody for 1 hr at room temperature. Membranes were washed with Tris-buffered saline with 0.1% Tween-20 after each antibody incubation and developed by using the chemiluminescence system (ECL Advance; Amersham Bioscience, Piscataway, NJ, USA). Source of primary antibodies: anti-phospho-IRF-3, anti-TLR3 and anti-Cleaved Caspase 3 from Cell Signaling (Beverly, MA, USA), anti-NOXA from Enzo Life Science (Lausen, Switzerland) and β-actin antibodies from Sigma-Aldrich. Horseradish peroxidase-conjugated goat antimouse or anti-rabbit Secondary antibodies were from Bio-Rad (Hercules, CA, USA).

The sample from brain tissues were separated by SDS-PAGE and transferred to PVDF filter (0.45 μm, millipore, USA). The membrane was incubated overnight at 4°C with primary antibodies including T-NF-κBp65/p-NF-κBp65(Ser536)/T-IκBa/p-IκBa/T-CREB/ p-CREB/ GAPDH (1:1000, Cell signaling Technology, USA), BDNF/Iba-1 (1:500, Abcam' RabMAb® technology), followed by incubation with appropriate HRP-conjugated secondary antibodies and finally visualized by chemiluminescence (ECL Advance; Amersham Biosciences), and exposure to X-ray films for 10 s −5 min.

Purified membrane fractions obtained after successive centrifugations of homogenized intestinal LMMPs preparations were used to analyze OTX1 and OTX2 protein level as described elsewhere (Giaroni et al., 2011 (link)). Membrane incubation with OTX1 and OTX2 primary antibodies was performed by incubation with a horseradish peroxidase-conjugated secondary antisera (Table 1). The signal of antibody/substrate complex was visualized by chemiluminescence using an enhanced chemiluminescence kit (ECL advance Amersham Pharmacia Biotech, Cologno Monzese, Italy), and then evaluated by densitometric analysis using Image J NIH image software. The effect of DNBS-induced colitis on OTX1 and OTX2 protein levels was expressed as the percentage variation of the optical density (expressed in arbitrary units) of OTX1 and OTX2 signals normalized to the respective β-actin, used as protein loading control, in LMMP preparations obtained from DNBS-treated animals compared to controls. Experiments were performed at least five times for each experimental group. Specificity of OTX1 and OTX2 primary antibodies was evaluated by testing their selectivity in spontaneous immortalized human Müller cell line (MIO-M1) and in rat hippocampus, respectively (data not shown) (Filpa et al., 2017a (link)). Negative controls were performed by omitting the primary antibody.

Hippocampus, prefrontal cortex, small intestine mucosa and mucosa-deprived whole wall samples were used to analyze BDNF and TrkB protein levels. Briefly, purified membrane fractions were obtained after successive centrifugations, boiled for 5 min in Laemmli sample buffer and processed for electrophoretic separation and blotting as described by Filpa et al. [37 (link)]. Membrane incubation with BDNF or TrkB primary antibodies was followed by incubation with a horseradish peroxidase-conjugated secondary antiserum (Table 1). The antibody/substrate complex was visualized by chemiluminescence using an enhanced chemiluminescence kit (ECL advance Amersham Pharmacia Biotech, Cologno Monzese, Italy). Signal intensity was evaluated by densitometric analysis using Image J NIH image software. β-actin and α-tubulin were used as protein loading control for intestinal and CNS areas, respectively. BDNF or TrkB protein levels were expressed as the ratio of the optical density (expressed in arbitrary units) of BDNF and TrkB bands and the optical density of the relevant β-actin or α-tubulin band x1000 obtained in CTR and ABX-treated samples. Experiments were performed at least five times. Negative controls were performed by omitting the primary antibody.

Colonic LMMPs preparations were used to analyze HAS2 protein level according to the method described by Giaroni et al.^{45 (link)}. Briefly, purified membrane fractions were obtained after successive centrifugations and boiled for 2 min in Laemmli sample buffer and processed as described elsewhere^{46 (link)}. Membrane incubation with HAS2 primary antibody, was followed by incubation with a horseradish peroxidase-conjugated secondary antisera (Table 1). The antibody/substrate complex was visualized by chemiluminescence using an enhanced chemiluminescence kit (ECL advance Amersham Pharmacia Biotech, Cologno Monzese, Italy). Signal intensity was evaluated by densitometric analysis using Image J NIH image software. β-actin was used as protein loading control. The effect of DNBS-induced colitis on HAS2 protein levels was expressed as the percentage variation of the optical density (expressed in arbitrary units) of HAS2 signal normalized to the respective β-actin in DNBS treated LMMPs compared to controls. Experiments were performed in quadruplicates. The specificity of HAS2 antibody was evaluated by testing its selectivity in NIH3T3 cells (data not shown). Negative controls were performed by omitting the primary antibody.

U251-MG cells were plated in 60 mm dishes and were treated with drugs. After 24 h, cells were washed with PBS 1× and centrifuged and lysed with RIPA buffer (Tris–HCl 50 mM pH 7.5, NaCl 150 mM, Triton-X 1%, SDS-20 0.1%, Na deoxycholate 1%, protease Inhibitor mix 1× ). Total cell lysates were quantified and equal amounts were electrophoresed on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane.
Proteins were identified by probing the membrane with the mouse anti-GFAP (Cell Signaling), anti-alphaB-Crystallin (Cell Signaling), and anti-tubulin antibodies (Sigma-Aldrich). Signals were detected using the chemiluminescence reagent ECL advance (Amersham) and protein levels in each sample were evaluated by comparison with corresponding amounts of the housekeeping tubulin. Densitometric analysis of proteins was performed by ImageJ software. For each sample, GFAP and anti-alphaB-crystallin amount was evaluated following normalization on the housekeeping tubulin.