Totalchrom software

Freeze dried mycelia were subjected to three cycles of freezing (-20°C) and thawing (room temperature) in 5% PCA. After the final thaw, samples were vortexed for 2 min and centrifuged for 8 min at 14,000 ×g. PAs and AAs were simultaneously dansylated and quantified using an HPLC method from Minocha and Long (2004) (link) with following modifications. Samples were incubated at 60°C for 30 min, cooled for 3 min and then microfuged at 14,000 ×g for 30 s. The reaction was terminated by the addition of 45 μl of glacial acetic acid. Sample tubes were kept open for 3 min under a flow hood to allow CO₂ bubbles to escape. Acetone used to dissolve dansyl chloride was evaporated using a SpeedVac Evaporator (Savant, Farmingdale, NY, United States) for 5 min. Finally, 1735 μl of filtered HPLC grade methanol was added to all tubes bringing the total volume to 2 ml. PAs and AAs were analyzed by HPLC method according to Minocha and Long (2004) (link). The data were processed using Perkin Elmer TotalChrom software (version 6.2.1).

RAW 246.7 cells were pretreated with BMP4 for 1 h and then incubated for 48 h in the presence of ox-LDL. After incubation, the cells were washed, thrice, with PBS and harvested in PBS. They were sonicated in an ice bath using an ultrasonic processor. The protein concentration of the lysate was then determined using the BCA assay with a portion of the lysate (100 μL) transferred to microfuge tubes. Then, stigmasterol (150 μL) was added to the rest of the lysate (450 μL) and mixed by vortexing. For the measurement of TC and FC, the mix was divided into two equal parts and KOH (100 μL 8.9 mol/L) was added to the TC sample, while the FC sample did not need to be saponified in KOH solution. After 2 h of incubation in a 50 °C water bath, 1 mL hexane was added to all tubes and subsequently centrifuged at 8000× g at 4 °C for 15 min. The supernatant of organic phases was collected and dried in a SpeedVac. The residues were collected and re-suspended in 400 μL acetonitrile:isopropanol (80:20, v/v) for further analysis. Then, the samples was injected in the HPLC system separately and analyzed with a System Chromatographer (PerkinElmer Inc., Waltham, MA, USA). Total Chrom software (PerkinElmer Inc.) was used to analyze the data. At the end, CE values were obtained by subtracting the FC values from the TC values. CE/TC was calculated using the following formula: (TC − FC)/TC × 100%.

Extracted wheat proteins from the grain and malt were analyzed according to the method of Wieser et al. [36 (link)] using high-performance liquid chromatography (HPLC) (Perkin Elmer Instruments, USA) coupled with Total-Chrom software and a photodiode array detector. Elution of AG, GLI and GLU was performed with linear gradient of acetonitrile (ACN/0.1%TFA) in the water (H₂O/0.1%TFA) from 24–54% over 30 min at flow rate of 1 mL min⁻¹ and column temperature of 50 °C. Proteins were separated on a C18 reverse phase column (5 μm, 4.6 × 150 mm; Sigma-Aldrich Chemie GmbH, Germany). Quantification of protein fractions was based on measuring its peak area at 210 nm. All the determinations were repeated twice. The peak areas under AG, GLI and GLU chromatograms were summed and used as a direct measure of total content of extractable wheat proteins. Consequently, the proportions (%) of protein fractions and single protein types were calculated.