The gel images from both the LF and HF groups were scanned on a Molecular Imager Pharos FX system (Bio-Rad, Hercules, CA, USA). Image analysis including image editing, spot finding, quantitation, and matching, was carried out using Quantity One Software (Bio-Rad) and PD Quest version 8.0 2-D gel analysis software (Bio-Rad, Hercules, CA, USA). The protein spots were detected by following the PD Quest software instructions using the following parameters: horizontal and vertical streaking removal with a radius of 33, smoothing by Power Mean filter with kernel size 3 × 3, speckle removal at a sensitivity of 50. The densities of protein spots were normalized using the Local Regression Model. The spots were then quantitatively compared between LF and HF groups using the approach by Sun et al.43 (link). Protein spots were considered to be differentially expressed if the difference between the average of spot densities between the two groups was twofold greater or lesser.
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