Imagemaster 2d platinum 6

Gels were scanned at a resolution of 300 dpi using a GS-800 calibrated densitometer (Bio-Rad Laboratories). The gel images were analyzed by Image Master 2D Platinum 6.0 software (GE Healthcare, Sweden). The software was used to automate the process of detecting and matching protein spots between the images. The significant expression changes (P<0.05) were determined using statistical tests such as student’s t-test on % volume of matched spots between the groups. Among the selected spots, fold change >1.3 and coefficient of variation <20 were considered for each spot.

The isoelectric focusing (IEF) was carried out using Ettan IPGphor 3 system (GE Healthcare, Piscataway, NJ, USA). The protein loading volume was adjusted to 0.6 mg/ml with IEF sample loading solution (7 M urea, 2 M thiourea, 2% CHAPS, 40 mM DTT, 0.5% IPG buffer, 0.002% bromophenol blue). Two-DE was performed as reported previously [32] . The gel images were acquired using ImageScanner III (GE healthcare, Piscataway, NJ, USA) and analyzed with ImageMaster 2D Platinum 6.0 (GE healthcare, Piscataway, NJ, USA). Triplicate runs were conducted for each sample to ensure reproducibility. For comparative analysis, the percentage intensity volume (%vol) of each spot was used for comparison of matched spots between serum-treated and untreated samples. To reduce potential errors, a ratio of ≥2 (or ≤0.5) and analysis of variance (ANOVA) <0.05 were taken as a threshold for differential expression. The differentially expressed protein spots were picked from the gels and washed first with water and then with 25 mM ammonium bicarbonate in 50% acetonitrile for 60 min. The gel spots were processed by enzymatic digestion and analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry as reported previously [32] .

From each sample, 25 μg of protein was loaded into 12% SDS-PAGE gels and then transferred onto a polyvinylidene fluoride (polyvinylidene difluoride) membrane (Millipore, USA). Anti-SPD_1495 antibody was incubated with polyvinylidene difluoride membrane at 4°C, and horseradish peroxidase-conjugated goat anti-mouse was used as a secondary antibody. The results were visualized with Clarity Western ECL Substrate (Bio-Rad, USA) and captured using ImageMaster 2D Platinum 6.0 (GE Healthcare, USA). Meanwhile, SDS-PAGE gels stained with Coomassie brilliant blue G250 were used as a loading control.

For proteomic data, statistical analysis was performed using t tests (p<0.05) available through the ImageMaster 2D Platinum 6.0 software (GE Healthcare, Uppsala, Sweden). Only proteins with significantly altered levels were excised for identification by MS.

PsaA, PiuA, and PiaA antibodies were derived from the previous work (Yang et al., 2016 (link)). The purified SPD_0090 protein was used as an antigen for the immunization experiments to generate multiclonal antibodies in mice according to the previous report (Brown et al., 2001b (link)). Equal amounts of proteins were analyzed by 10–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, United States), which were incubated with the corresponding antibodies at 4°C. This was followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse as a secondary antibody. Results were visualized with Clarity Western ECL substrate (Bio-Rad, United States) and captured using Image Master 2D Platinum 6.0 (GE Healthcare, United States).

The patterns of spots on the 2-DE gels were analyzed using the gel image analysis software PDQuest™ (Bio-Rad Laboratories, Inc.) or Image Master 2D Platinum 6.0 (GE Healthcare).