Gbox chemi xt4 system

Total protein of 200 mg of grinded mycelia was extracted as described by Apostolaki et al. [56 (link)] and protein concentration was determined by the Bradford assay. Total proteins (50 µg) were separated by SDS-PAGE (10% (w/v) polyacrylamide gel) and electroblotted (OmniPAGE Electroblotting Units, Cleaver Scientific Ltd) onto a nitrocellulose membrane (0.45 µm, Thermo Scientific). Membrane was then incubated in stripping buffer (10 mM Tris–HCl pH 6.8, 2% SDS and 100 mM β-mercaptoethanol) for 15 min at 55°C (as described by Kaur & Bachhawat [57 (link)]). Membrane was then treated with 5% non-fat dry milk, and immunodetection was done with a primary mouse anti-GFP monoclonal antibody (Roche Applied Science), or a mouse anti-actin monoclonal (C4) antibody (MP Biomedicals) and a secondary sheep anti-mouse IgG HRP-linked antibody (GE Healthcare). Blots were developed using the Amersham ECL Western blotting detection reagents and analysis system (GE Healthcare), and images were acquired using the GeneSys software from the GBoxChemi XT4 System (Syngene).

RAW264.7 cells were washed with PBS and lysed in an ice-cold radio immunoprecipitation buffer. Protein (50 μg) were electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene difluoride membrane. The blocked membranes were incubated with the primary antibodies against iNOS, COX-2, NF-κB, and IκBα and subsequently incubated with second antibody. Protein density was quantitated with G BOX Chemi XT4 system (SYNGENE, Cambridge, UK). The basal levels of the proteins were normalized by analyzing the level of β-actin protein.

The protein was extracted from liver tissues. BCA protein assay kit (CoWin Bioscience, Beijing, China) was applied to determine protein concentration. The protein (100 μg for each sample) was separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blockade with 5% skim milk, the membrane was incubated with targeted primary antibodies overnight at 4°C and then with secondary antibody at room temperature for 1 h. The Phospho-AKT and β-actin antibodies and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Finally, the signals were detected by enhanced chemiluminescence (ECL) Detection Kit (Millipore, Billerica, MA, USA) and captured by G: BOX Chemi XT4 System (Syngene, Cambridge, UK). GeneTools software (Syngene) was used for quantification. The relative protein expression was normalized to β-actin.

Images of whole larval bodies (16-bit grayscale) were acquired with G:BOX Chemi XT4 system (Syngene, Cambridge, UK) and analyzed using ImageJ software (NIH, Bethesda, MD, USA). Larvae have been immobilized by chilling them on ice for 5 min then the pictures have been taken in uniform white led light and constant acquisition parameters.

Following electrophoresis, proteins were transferred to PVDF membrane (Immobilon-P, IPVH00010, Millipore) in a Trans-Blot Turbo transfer system (Bio-Rad) for 30 min at 25 V. The PVDF membrane was blocked in a solution of 5% non-fat milk powder in TBS-T (50 mM Tris pH 7.6, 150 mM NaCl, 5% milk, 0.05% Tween-20) for 1 h at room temperature. All primary antibody hybridisations were conducted overnight at 4°C, whereas secondary or tertiary antibody hybridisations were performed at room temperature for an hour. Antibodies are given in Supplementary Table 1 and were used at the dilutions recommended by the manufacturer. After each hybridisation, membranes were washed with TBS-T, with changes every 5 min for 1 h. Membranes were developed by incubation with enhanced chemiluminescence substrate (Thermo Scientific) for 2 min at room temperature and imaged using a G:BOX ChemiXT4 system (Syngene).

Cardiac fibroblasts were treated for 10 min (for p38α phosphorylation) or 6 h (for TNC expression) with Yoda1 (10 μM) or its vehicle (DMSO) and lysed in a buffer containing: 10 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, protease inhibitors (Roche, Basel, Switzerland), and phosphatase inhibitors (Roche, Basel, Switzerland). A total of 25 μg of protein extracts were loaded on 10% polyacrylamide precast gel (Bio-Rad, Hercules, CA, USA). Proteins were transferred onto PVDF membranes, blocked in milk (5%) for 1 h before incubation with the primary antibody against phospho-p38 MAPK (9215, Cell Signalling Technology, Danvers, MA, USA; 1:250), or reprobed for p38α antibody (9228, Cell Signalling Technology, Danvers, MA, USA; 1:500) overnight at 4 °C. For TNC, membranes were incubated with primary antibody against TNC (JP10337, Tecan, Männedorf, Switzerland; 1:200) or reprobed for α-tubulin antibody (3873, Cell Signalling Technology, Danvers, MA, USA; 1:2000). Membranes were then washed and incubated with anti-mouse and anti-rabbit secondary antibodies (GE Healthcare, Chicago, IL, USA; 1:5000). Visualization was performed using ECL detection reagent and Syngene G:BOX Chemi XT4 system. Blots were analysed with ImageJ software (v1.52p, Bethesda, MD, USA).

Protein concentration was determined using a BCA protein assay kit (CoWin Bioscience, Beijing, China). The protein sample was resolved by 10% denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis and was blotted onto Immobilon-P PVDF membranes (Millipore, Billerica, MA, USA). After being blocked by 5% milk, the membrane was incubated with primary antibodies at 4°C overnight. The antibodies against c-Jun N-terminal kinase (JNK), Phospho-JNK, activated Caspase-3, Bcl-2-associated X (Bax) protein, and Bcl-2 were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against CYP2E1 was obtained from Abcam (Cambridge, MA, USA), and β-actin was obtained from Huaan Biological Technology (Hangzhou, China). Subsequently, the membrane was incubated in a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody from Thermo Scientific (Rockford, IL, USA) for 1 h. The signal was visualized by enhanced chemiluminescence HRP substrate (Millipore, Billerica, MA, USA) and acquired by GBOX Chemi XT4 System (Syngene, Cambridge, UK). GeneTools software (Syngene) was used for quantification.