Trans blot turbo midi nitrocellulose transfer packs

The native antigens separated from the bovine hydatid cyst fluid (BHCF) were analysed via SDS-PAGE and Western blotting according to [33 (link)]. Briefly, each lane of a 12% w/v polyacrylamide gel was loaded with 30 μL native antigen (11.65 μ g/μL), protein-loading (Laemmli) buffer and protein marker (PageRuler^Tm Cat# 26614, thermo scientific), separated by 12% w/v polyacrylamide gel and transferred to the nitrocellulose membrane (NCM, 0.22 µm, Trans-Blot^® Turbo™ Midi nitrocellulose transfer packs # 1704159, Bio-Rad, USA) using the Trans-Blot^® Turbo™ transfer system (Bio-Rad, USA). The blots were cut into strips, labelled and blocked with (5% w/v) skimmed milk in tris buffer saline (20 mM Tris-HCL, pH 7.2, 150 mM NaCl). The positive serum collected from infected tagged animals (three buffalo, each with 2–3 hydatid cysts in the liver, confirmed after post-mortem) was used as the primary antibody. The strips were charged with the anti-bovine antibody (Anti-bovine IgG-alkaline phosphatase conjugate Sigma Cat # A0705-25ML) as the secondary antibody (1:20,000) after three washings. A chromogenic substrate (NBT/BCIP bio-WORLD, Dublin, OH, USA) was used as a colour marker.

COS-7 cells were transfected with 70+TRPM1, 92+TRPM1, 109+TRPM1 plasmids or not transfected. After 48 hours, cells were dissociated by sonication in lysis buffer (Tris 50mM pH7.5, NaCl 150 mM, Triton X-100 1%) along with a combination of anti-protease and anti-phosphatase (Phosphatase Inhibitor Cocktail, Sigma-Aldrich) on ice for 30 minutes. Lysates were cleared by centrifugation at 13,800 x g for 10 minutes at 4°C. Protein extracts from transfected and untransfected cells were separated by SDS-Page on a 4–15% pre-cast gel (Mini-PROTEAN^® TGX^™ Precast Protein Gels, Bio-Rad, Hercules, CA, USA) and subsequently transferred on nitrocellulose membranes (Trans-Blot^® Turbo^™ Midi Nitrocellulose Transfer Packs, Bio-Rad). Membranes were blocked in milk for an hour and then incubated overnight at 4°C with the patients’ sera (1:5,000–1:50,000) or serum from a non-MAR patient (1:50,000) and anti-V5 antibody (1:5,000, Invitrogen, ThermoFisher Scientific). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated donkey anti-human or anti-mouse IgG (1:10,000, Jackson ImmunoResearch) as secondary antibody for 1 hour at room temperature. Bands were revealed using an HRP substrate for chemiluminescence (Pierce^™ ECL Western Blotting Substrate, ThermoFischer).

The Western blot analysis was performed as previously described [42 (link)]. In brief, cells were lysed with lysis buffer (50 mM Tris; pH 7.6; 150 mM NaCl; 5 mM EDTA; 1 mM Na₃VO₄; 10 mM NaF; 10 mM sodium pyrophosphate; 1% NP-40) containing protease inhibitor cocktail (0.01 M EDTA; 1 mM DTT; 1 mM Leupeptin; 1 mM PMSF; and 1 µM Aprotinin). The cell lysate was placed on ice for 60 min and centrifuged at 17,982× g for 15 min. The total protein was quantified using the Bradford method (Quick Start ^TM Bradford Protein Assay; Bio-Rad, Hercules, CA, USA). Total proteins were separated by 12% polyacrylamide gel and transferred onto Trans-Blot Turbo Midi Nitrocellulose Transfer Packs (Bio-Rad, Hercules, CA, USA). The membrane was incubated with primary followed by secondary antibodies after blocking with 5% non-fat milk. Immunodetection was performed using the ECL KIT (Amersham Biosciences, Uppsala, Uppland, Sweden) in ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and quantified by the platform for scientific image analysis ImageJ (NIH) [46 (link)]. The following antibodies were used: polyclonal anti-MTAP antibody (Proteintech, Rosemont, IL, USA) diluted 1:800 and anti-β actin antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1:2000.

The western blot analysis was performed as previously described [60 (link)]. In brief, cells were lysed with Cell Lysis Buffer (Cell Signaling) containing Protease Inhibitor Cocktail (Sigma). Total proteins were separated by a 4–20% Criterion TGX Precast Gel (Bio-rad) and then transferred onto Trans-Blot® Turbo™ Midi Nitrocellulose Transfer Packs (Bio-rad). The membrane was incubated with primary followed by secondary antibodies after blocking with 5% non-fat milk (Bio-rad). Immunochemical detection was performed using either the Thermo Scientific™ SuperSignal™ West Femto Chemiluminescent Substrate (Thermo Scientific) or Amersham™ ECL™ Western Blotting Detection Reagent (GE Healthcare Life Science). The following antibodies were used at the dilution recommended by the manufacturer: β-actin (Sigma Aldrich, AC-15), GAPDH (Santa Cruz, sc-51905), XIAP (Cell Signaling, #2042), PARP (Cell Signaling, #9542), Survivin (Cell Signaling, 6E4), c-IAP (Cell Signaling, #4952), ZEB1 (Santa Cruz, sc-10572), and vinculin (Cell Signaling, E1E9V).

Cells were harvested in exponential phase at an OD₆₀₀ of 0.8, as previously described [29 ]. A total of 6.0 OD₆₀₀ cells were then lysed in 0.2 M NaOH, supplemented by complete™ Protease Inhibitor Cocktail (Roche) and Halt™ Phosphatase Inhibitor Cocktail (Fischer scientific), incubated for 20 min on ice, pelleted, resuspended in 50–70 μl sample buffer (1 × Laemmli buffer (80 mM Tris–HCL pH 6.8, 10 mM EDTA pH 8.0, 8% SDS, 20% Glycerol, 0.004% Bromophenol blue), 8 M Urea, 2.5% β-Mercaptoethanol) and heated for 5.0 min at 90 °C. About 15 μl of the protein sample was loaded per lane of a 12% Criterion TGX Precast Midi Protein Gel (Bio-Rad, Hercules, CA, USA), blotted onto Trans-Blot Turbo Midi Nitrocellulose Transfer Packs (Bio-Rad, Hercules, CA, USA), and probed overnight with primary antibodies: Membranes were probed with anti-GFP (ab6556; Abcam). As secondary antibodies, goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680 (LI-COR; 1/20000 dilution) were used. Membranes were scanned using the LI-COR Odyssey Infrared scanner. Images were then quantified by the ImageJ software.

Leaf samples from wild-type (WT) plants and plants from line 131 were harvested 72 h after P. pachyrhizi inoculation or mock treatment. Proteins were extracted from four foliar discs of the same soybean plant with 250 μl of extraction buffer (Tris-Hcl 100 mM, NaCl 100 mM, DTT 0.04%) and placed on ice for 10 min before centrifugation at 4 °C for 10 min. The protein concentration was determined with the Bradford method using the Bio-Rad Protein assay dye reagent solution. For denaturation, 1 volume of Laemmli buffer (Bio-Rad, US) was added to 1 volume of extracted proteins (30 μg). The mixture was kept for 5 min at 95 °C and 5 min on ice before loading on a TGX 4–20% Stainfree (Bio-Rad US) gel immersed in TGS 1X buffer. After migration, separated proteins were transferred onto a membrane by using Trans-Blot® Turbo™ Midi Nitrocellulose Transfer Packs (Bio-Rad) and the TransBlot Turbo device (Bio-Rad, US). Membrane blocking and incubation with the antibodies were performed as suggested by the provider. GFP antibodies (Sigma) and Immun-Star Goat Anti-Rabbit (GAR)-HRP Conjugate antibody were used. Antibody detection was realized with the Clarity™ Western ECL (Bio-Rad, US) kit following the supplier’s instructions. Finally, the ChemiDoc™ Touch camera (Bio-Rad, US) was used to record the results.