Immobilon reagent

Plasma samples were collected and subjected to western blotting. Briefly, 10 μL plasma samples were fractionated on 10% SDS-polyacrylamide gels and then transferred to Sequi-Blot polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with each primary antibody (Ab) (anti-SFRP4 1 : 1,000 and anti-β-actin 1 : 1000) at 4°C overnight, as recommended in the manufacturer's instructions. After washing 3 times, they were incubated with horseradish peroxidase conjugated secondary Ab for 2 hr. Signals were detected using the Immobilon reagent (Millipore, Billerica, MA, USA) and visualized using an LAS-400 Lumino Image Analyzer (Fujifilm, Co., Tokyo). Visualized signal intensities were quantitatively analyzed using MultiGauge software (Bio-Rad, Hercules, CA, USA). All primary Abs were purchased from Cell Signaling Technology (Beverly, MA, USA).

Fresh liver samples were homogenized in lysis buffer at 4°C followed by centrifugation at 12,000 rpm at 4°C for 10 min to remove debris. The resultant supernatants were collected and subjected to Western blotting. Briefly, 20 μg lysates were fractionated on 10% SDS-polyacrylamide gels and then transferred to Sequi-Blot polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with each primary antibody (Ab) (anti-XBP1 1 : 1000, anti-BiP 1 : 1000, anti-p-PERK 1 : 1000, and β-actin 1 : 1000) at 4°C overnight, as recommended in the manufacturer's instructions. After washing 3 times, they were incubated with horseradish peroxidase-conjugated secondary Ab for 2 hr. Signals were detected using the Immobilon reagent (Millipore, Billerica, MA, USA) and visualized using an LAS-400 Lumino Image Analyzer (Fujifilm, Co., Tokyo). Visualized signal intensities were quantitatively analyzed using MultiGauge software (Bio-Rad, Hercules, CA, USA). All primary Abs were purchased from Cell Signaling Technology (Beverly, MA, USA).

Cells were lysed with lysis buffer (4 °C) containing 20 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 50 mM NaCl, 1 mM EGTA, phosphatase inhibitors 2 and 3, and a protease inhibitor mixture (Sigma). γ-tubulin was immunoprecipitated using mouse monoclonal antibody followed by addition of protein G-agarose beads. The beads were washed with lysis buffer and then boiled in SDS-PAGE sample buffer for immunoblot analysis. Membranes were developed for immunoblot using the Immobilon reagent (Millipore), followed by imaging using ChemiDoc XRS System (Bio-Rad). Ser 558-phosphorylated TACC3 was probed by rabbit polyclonal phospho-TACC3 antibody (Cell Signaling, USA).

Western blotting was performed as previously described 17 . Briefly, cells were lysed and separated on 10% SDS-polyacrylamide gels. The following primary antibodies (Abs) were used: GINS2, B-cell lymphoma-2 (Bcl-2), Bax, caspase-3, cyclin dependent kinase inhibitor 1a (CDKN1A), CDKN1B, cyclin dependent kinases 4 (CDK4), cyclin D, bone morphogenetic protein receptor type 2 (BMPR2), interferon induced protein with tetratricopeptide repeats 1 (IFIT1), STAT1, STAT2, interferon regulatory factor 1 (IRF-1), tumor necrosis factor alpha induced protein 3 (TNFAIP3), interferon induced transmembrane protein 1 (IFITM1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Signals were detected using the Immobilon reagent (Millipore, USA) and visualized using a Bio-Rad ChemiDoc XRS (Bio-Rad, USA). Visualized signal intensities were quantitatively analyzed using Quantity One software (Bio-Rad, USA). All primary Abs were purchased from Cell Signaling Technology (Beverly, USA).

HUVECs were lysed in RIPA lysis buffer (Beyotime, Jiangsu, China), the cellular debris were centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was used for western blotting analysis. We transferred equal amounts of protein extract that were separated by SDS-PAGE (10%) onto polyvinylidene fluoride (PVDF) membranes and then probed with primary antibodies of maspin (Abgent, USA) and MMP2 (BD Pharmingen, San Jose, CA, USA) both at a dilution of 1:100. After incubation with primary antibodies at 4 °C overnight, the blots were probed with horseradish peroxidase-conjugated secondary antibody (Abgent, USA) at a 1:3000 dilution for 30 min at room temperature. The signals were detected by Immobilon reagent (Millipore, Billerica, MA) and visualized by image analyzer (Bio-Rad, Hercules, CA, USA).

Colonic specimens were dissected to separate the mucosal/submucosal layer from underlying neuromuscular tissues. Samples of colonic muscular tissue or ICSMCs were homogenized in RIPA lysis buffer (Cole Parmer homogenizer, Generalcontrol SpA, Milano, Italy). Homogenates were spun by centrifugation at 20,000 × g for 15 min. at 4°C. Supernatants were then separated from pellets and stored at −80°C. Protein concentration was determined by the Bradford method (Protein Assay Kit; Bio-Rad, Hercules, CA, USA). Equivalent amounts of protein lysates (50 μg for tissues and 10 μg for ICSMCs) were separated by 8% SDS-PAGE for immunoblotting. After transfer onto a PVDF membrane, the blots were blocked and incubated overnight with a rabbit anti-collagen I antibody (Ab34710; Abcam, Cambridge, UK) or a goat anti-transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) antibody (Table1). After repeated washings with TBS-T, appropriate secondary peroxidase-conjugated antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA) were added for 1 hr at room temperature. Immunoreactive bands were then visualized by incubation with chemiluminescent reagents (Immobilon reagent; Millipore, Billerica, MA, USA), and examined by Kodak Image Station 440 for signal detection. To ensure equal sample loading, blots were stripped and reprobed for determination of β-actin by a specific antibody (P5747; Sigma-Aldrich, Milan, Italy).

Primary B cells before and 2 dpi or 5 dpi after EBV infection as well as EBV-immortalized B lymphocytes treated with either 25-HC (2 µM) or the solvent ethanol for 48 h were lysed in RIPA buffer containing protease inhibitors (Roche cOmplete), mixed with 5× sample buffer before being separated on a denaturing 7.5% polyacrylamide gel by discontinuous electrophoresis (SDS-PAGE), followed by semidry blotting onto nitrocellulose membrane. The blot was probed with primary mouse antibodies against the human ABCD1 protein (Euromedex ALD-1D6-AS, clone 2AL-1D6, 1:10,000) and β-actin (#603024087, Chemicon, 1:100,000) followed by a goat anti-mouse secondary antibody conjugated with horseradish peroxidase (#P0447, Dako, 1:30,000). For detection, the Immobilon Western HRP Substrate Peroxide Solution and Immobilon Reagent (Millipore) were used with the ChemiDoc Imaging System and Image Lab software (Bio-Rad).