Immobilon western

For Western blots, whole-cell protein extracts were prepared from peripheral blood mononuclear cells (PBMCs) by using radio immunoprecipitation assay (RIPA) lysis buffer (Thermo Scientific, Rockford, USA). Protein concentrations were determined using the BCA Protein Assay Kit (Pierce, Thermo Scientific, Rockford, USA). An equal amount of 30 μg of protein for each sample was separated by electrophoresis on 12% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes using the iBlot™ DryBlotting device and iBlot™ Transfer stacks. The blots were incubated with the rabbit monoclonal anti-GM-CSF receptor alpha primary antibody at 1:1,000 (Abcam, Cambridge, MA, USA) overnight at 4 °C. Labelling of the first antibodies was detected using relevant secondary antibodies conjugated to horseradish peroxidase (HRP) (1:10,000; Merck Millipore) and detected using a chemiluminescent HRP substrate kit (Immobilon Western; Merck Millipore). The protein bands were detected and scanned by a gel image system (Top Bio Co., MultiGel-21).

The membrane obtained after the preceding step was washed twice. The chemiluminescence signal was detected using a Chemi-Stage CC16mini (KURABO, Osaka, Japan) after development with Novex AP chemiluminescent substrate (Thermo Fishes Scientific, Waltham, MA). For the scFv-library, the membrane was incubated for 2 h with a horseradish peroxidase (HRP)-conjugated anti-His antibody (1:5000 in PBS-T; FUJIFILM Wako Pure Chemicals), and then washed extensively in PBS-T. The chemiluminescence signal was detected using a Chemi-Stage CC16mini after development with a chemiluminescent HRP substrate kit (Immobilon Western; Merck Millipore). The filter harboring the colonies and the image presenting the chemiluminescence results were superimposed, allowing the positive colonies corresponding to the chemiluminescence signals to be identified.

Cells were lysed in lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific), and the debris were removed by centrifugation at 10,000 × g and 4 °C. Protein concentration was determined with a micro-BCA kit (Thermo Fisher Scientific). Samples were then boiled in SDS sample buffer (Novex) containing 10% β-mercaptoethanol (Sigma) and resolved by SDS–polyacrylamide gel electrophoresis. Immunoblot analysis was performed with specific antibodies and the antigen–antibody complexes were visualized by chemiluminescence (Immobilon Western, Merck Millipore).

To evaluate autophagic activity, whole-cell proteins were extracted from macrophages treated with low (10 µg/mL) and high (100 µg/mL) concentrations of FLG for 24 h along with untreated and LPS-treated cells using Laemmli buffer (Tris-HCl 125 mM pH 6.8, 2% (w/v) sodium dodecyl sulfate (SDS); 10% (v/v) glycerol; 5% (v/v) β-mercaptoethanol). Cell lysates were separated on a 4–20% gradient gel (Biorad, Schiltigheim, France) and then transferred onto a polyvinylidene difluoride (PVDF) membrane. Cell membranes were blocked with PBS containing 0.1% (v/v) Tween 20 (PBS-T) and 5% (w/w) non-fat dry milk for 1 h and incubated 50 min at 4 °C with a 1 µg/mL anti-LC3 antibody in PBS-T containing 5% non-fat dry milk. The antibodies used for western immunoblotting were specific for the LC3 autophagic marker (MBL, clone 51-11, #M115-3) and the β-actin loading control (C4 mouse monoclonal antibody, Santa Cruz Biotechnology, #47778). After washing with PBS-T, the membranes were incubated for 30 min at room temperature with goat anti-mouse IgG antibody (Southern Biotech, Birmingham, Alabama, #1030-05) conjugated to horseradish peroxidase (HRP). The signal was detected using enhanced chemiluminescence detection reagents (Immobilon Western, Merck Millipore, Darmstadt, Germany, #WBKLS0500) and visualized on radiographic film in a Kodak processor (#M35-M X-OMAT, Rochester, NY, USA).

Proteins were isolated from Caco-2 cells using RIPA buffer and subsequently subjected to Western blot analysis as described by Dinić et al. (2017) (link). Briefly, the extracted proteins (10 μg) were separated on 12% SDS–PAGE and transferred to 0.2 mm nitrocellulose membrane (GE Healthcare, Chicago, IL, United States) using Bio-Rad Mini trans-blot system (Bio-Rad, Hercules, CA, United States). The membranes were incubated for 2 h with anti-claudin (CLDN-4) antibody (1:1000; Novus Biologicals, United States) and anti-β-actin (1:1000; Thermo Fisher Scientific). The membranes were washed and incubated with appropriate HPR-conjugated secondary antibodies (goat anti-rabbit; 1:10000; Thermo Fisher Scientific) for 1 h at room temperature. Proteins were detected by enhanced chemiluminescence (Immobilon Western, Merck Millipore).

Lysates were prepared from frozen tissue samples and cultured cells using the lysis buffer containing 50 mm HEPES (pH 7.4), 1% Triton X-100, 50 mm sodium pyrophosphate, 0.1 m sodium fluoride, 10 mm EDTA, 10 mm sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mm benzamidine, and 2 mm phenylmethylsulfonyl fluoride. After protein electrophoresis and transfer, immunoblots were performed using rabbit anti-serum as primary antibody at a 1:1,000 dilution. The blot was followed by a 1:10,000 dilution of goat anti-rabbit horseradish peroxidase-conjugated secondary antibody kit (Immobilon™ Western; EMD Millipore, Billerica, MA, USA) as previously described25 (link). GAPDH was used as a loading control. The maximum intensity of each band was quantified using ImageJ software. Ratios of P-AMPK/AMPK, P-ACC/ACC, and P-p46/p46 were normalized to GAPDH and adjusted relative to the average of PBS-treated control, which was arbitrarily set as 1 (AU).

Cell lysates and supernatants mixed with the sample buffer were incubated at 95 °C for 10 and 5 min, respectively. After 12% sodium dodecyl sulfate (SDS)-PAGE, separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The PVDF membranes were soaked with 3% skim milk (Becton Dickinson, Franklin Lakes, NJ, USA) in PBS and washed with PBST. Each membrane was incubated with a mouse anti-M1 MAb (APH 6-23-1-6) [34 (link)] and a mouse anti-beta actin antibody (ab6276, Abcam, Cambridge, UK) as primary antibodies and subsequently with HRP-conjugated goat anti-mouse IgG (H + L) (115-035-062, Jackson Immuno Research, West Grove, PA, USA) as a secondary antibody. These antibodies were diluted with PBST containing 1.5% skim milk. The bound antibodies were visualized with Immobilon Western (Merck Millipore, Darmstadt, Germany). The amount of the viral M1 protein was semi-quantified based on the band intensity using an Amersham Imager 600 (GE Healthcare, Little Chalfont, UK).