Trans blot turbotm

Prepared protein lysates were then electrophoresed on TGX Precast gels (Bio-Rad Laboratories), and proteins were transferred to PVDF membranes using a Trans-Blot^® Turbo^TM (Bio-Rad Laboratories). After washing, membranes were treated by PDVF Blocking Reagent^® (Toyobo Co., Ltd., Osaka, Japan), then incubated with a primary antibody diluted in Can Get Signal^® Solution-1 (Toyobo Co., Ltd.). After thorough washing with PBS containing 0.5% of Tween-20 (PBS-T), membranes were incubated with horse-radish peroxidase-conjugated secondary antibody in Can Get Signal Solution-2 (Toyobo Co., Ltd.), and washed with PBS-T. Chemiluminescence was produced using Luminata Forte (EMD Millipore Corporation, Billerica, MA, United States) and detected with LumiCube (Liponics, Tokyo, Japan). Primary antibodies used were anti-Nrf2 (1/1000 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), and anti-histone H3 (1/4000; Cell Signaling Technology Japan, Tokyo, Japan).

Bacillus isolate B29 strain integrated with recombinant CC chemokine protein was grown in LB broth containing 100 µg/mL of kanamycin at 37 °C with 180 rpm agitation for 18 h. Bacterial cell cultures were harvested by centrifugation at 5000 rpm for 10 min. The pellet was resuspended in 1 × phosphate-buffered saline (PBS, pH 7.4) and sonicated for 3 min in an ice bucket, with 2 s each. Western blot analyses were applied to confirm the existence of secreted Nile tilapia CC chemokine produced from Bacillus isolate B29. The recombinant CC chemokine protein was separated on SDS-PAGE gel and transferred onto a nitrocellulose membrane using a Trans-Blot^® Turbo^TM (Bio-Rad Laboratories, Hercules, CA, USA)) at 25 V, 1.0 A for 30 min. To minimize non-specific binding, the membrane was blocked with 2% skim milk at room temperature for 1 h. The Western blot assays were performed using anti-His tag mouse monoclonal antibody and goat anti-mouse IgG with horseradish peroxidase (HRP). The target proteins were detected using SuperKine™ West Pico PLUS Chemiluminescent Substrate (Abbkine Scientific Co., Ltd., Redlands, CA, USA) and visualized with enhanced chemiluminescence (ECL) solution using ChemiDoc MP Imaging System^TM (Bio-Rad Laboratories, Hercules, CA, USA).

For Western blot analysis 50 μg of protein was loaded onto a 12% polyacrylamide gel Mini‐ PROTEAN TGXTM (BIO‐RAD, Milan, Italy) followed by electrotransfer to nitrocellulose membrane Trans‐Blot TurboTM (BIO‐RAD, Mylan, Italy) using Trans‐Blot SE Semi‐Dry Transfer Cell (BIO‐RAD). Subsequently, membrane was blocked in chemiluminescent blocker (Millipore, Darmstadt, Germany) for 1 h at room temperature. After blocking, the membrane was three times washed in phosphate‐buffered saline (PBS) for 5 min and incubated with primary antibodies against human MCT1 (ab90582, Abcam, Mylan, Italy), MCT4 (ab234728, Abcam), and β‐actin (ab181602, Abcam). Next day, after three washes in TBST, the membranes were incubated with antimouse (1:3000, Jackson, WestGrove, PA) and anti‐rabbit HRP‐conjugated (1:3000, Jackson, WestGrove, PA) secondary antibodies for 1 h at RT. Proteins bands were visualized with premixed ready‐to‐use chemiluminescent HRP detection reagent (Millipore) according to the manufacturer's instructions and captured using the C‐DiGit Blot Scanner (LI‐COR Biosciences, NE). The density of each band was quantified using ImageJ analysis software and normalized β‐actin levels measured in the same membranes.

An extract of Alternaria alternata from strain 103.33 was obtained by double water-soluble extraction and subsequent freeze-drying.
Proteins from the A. alternata extract were analyzed by SDS-PAGE, according to the Laemmli method, in 15% polyacrylamide gels under reducing conditions. Proteins were visualized by silver staining (Thermo Scientific, Waltham, MA, USA) and transferred to polyvinylidene difluoride membranes (PVDF, Trans-blot turbo TM. Bio-Rad, Hercules, CA, USA).
The binding of IgE antibodies to allergens was analyzed by immunoblotting using individual patients’ sera and anti-human IgE peroxidase conjugate (Southern Biotech, Birmingham, AL, USA). Chemiluminescence detection reagents (Western lightning^® Plus-ECL, Perkin Elmer, Waltham, MA, USA) were added, following the manufacturer’s instructions.
The strength of IgE binding to the Alt a 1 protein was measured using Image Lab software version 5.2.1 build 11 (Bio-Rad, Hercules, CA, USA). In all cases, the relative intensity of the Alt a 1 band at baseline time in each patient was considered as a reference band and, therefore, as 100% IgE binding (relative quantification = 1). Finally, at successive times (Year 1 and Year 2), the intensity of the Alt a 1 band was compared with the intensity at baseline.

Briefly, for western blot analysis 50 μg of proteins were loaded onto a 12% polyacrylamide gel Mini- PROTEAN® TGXTM (BIO-RAD, Milan, Italy) followed by electrotransfer to nitrocellulose membrane Trans- Blot® TurboTM (BIO-RAD) using Trans- Blot® SE Semi-Dry Transfer Cell (BIO-RAD). Subsequently, membrane was blocked in Odyssey Blocking Buffer (Licor, Milan, Italy) for 1 h at room temperature. After blocking, membrane was washed three times in PBS for 5 min and incubated with primary antibodies against human MyD88, TLR4 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), overnight at 4 °C. Next day, membranes were washed three times in PBS for 5 min and incubated with Infrared anti-mouse IRDye800CW (1:5000) and anti-rabbit IRDye700CW secondary antibodies (1:5000) in PBS/0.5% Tween-20 for 1 h at room temperature. All antibodies were diluted in Odyssey Blocking Buffer. The blots were visualized using Odyssey Infrared Imaging Scanner (Licor, Milan, Italy) and protein levels were quantified by densitometric analysis of antibody responses. Data were normalized to protein levels of β-actin.

2 μg of recombinant purified rLpqH^His was reduced in 1X Laemmli buffer for 5 min at 95 °C. After electrophoresis (12% acrylamide SDS-PAGE), proteins were transferred to a nitrocellulose membrane (TransBlot^®-TurboTM, Bio-Rad). The membrane was blocked with 1% Bovine Serum Albumin (Sigma-Aldrich) in TBS buffer and incubated over night with the PhosphoDetect™ anti-phosphoserine (1C8, 4A3, 4A9, 16B4 Merck Millipore), anti-phosphothreonine (1E11, 4D11, and 14B3, Merck Millipore), and anti-His (Sigma-Aldrich) antibodies at 4 °C. After washing four times with TBST-buffer (TBS with 0.1% Tween20), the membrane was incubated at room temperature for 1 h with anti-mouse horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) diluted in TBST-buffer. The detected protein signals were visualized by an enhanced chemiluminescence reaction system using the Amersham Biosciences ECL kit and revealed with the ChemiDoc™ Touch Imaging System (BioRad).

The cells were lysed with 100 µL of lysis buffer (50 mM Tris-HCl, pH = 7.4), sodium dodecyl sulphate (SDS) 0.5%, 5 mM EDTA, 10 µL of protease inhibitors cocktail (PIC) (Millipore, cat# 539133) and phosphatase inhibitor cocktail (ThermoFisher Scientific, Waltham, MA, USA; cat# 78428) and collected in a 1.5 mL tube. The lysates were boiled at 96 °C for 5 min and then quantified with a QuantiPro BCA Assay Kit (Sigma-Aldrich, Milan, Italy; cat# SLBF3463). Then, 10 µg (for STIM1) and 20 µg (for ORAI1) of proteins were mixed with the right amount of Laemmli Sample Buffer 4X (Bio-Rad Laboratories), and boiled. The samples were then loaded onto a 12% polyacrylamide–sodium dodecyl sulphate gel for SDS-PAGE. The proteins were transferred onto nitrocellulose membrane, using Mini Transfer Packs with Trans-Blot^® Turbo TM (Bio-Rad Laboratories) according to manufacturer’s instructions (Bio-Rad Laboratories). The membranes were blocked in 5% skimmed milk (Sigma, cat# 70166) for 45 min at room temperature. Subsequently, the membranes were incubated with indicated primary antibody, overnight, at 4 °C. The primary antibodies used are listed in Table S3; anti-β-Actin was used to normalise the protein loading.