Pdquest advanced 2d gel analysis software

The image analysis was performed with PDQuest Advanced 2D Gel Analysis Software (Bio-Rad, Hercules, CA, USA). Spot detection was automatically performed using the software algorithm and the spots were verified manually. After the insertion of an appropriate number of user seeds, the matching was performed automatically and then checked manually. To ensure normalization of the spot quantities, the protein spot densities were normalized (%V) on total volumes of all the spots in each gel image.

Gel imaging and spot quantification were carried out using the PD-Quest Advanced 2D Gel Analysis software v8.0.1 (Bio-Rad) applying automatic spot detection and matching followed by manual/visual validation. The image was normalized by local regression and a scaling factor of 10⁶ (ppm) was applied to avoid non-expression related variations in spot intensity. Spots showing more than 2-fold changes in intensity with a coefficient of variation <50% and with statistically significant differences by Student’s t-test (P < 0.05) were defined as differentially expressed.

Fruit pericarp flesh was ground in liquid nitrogen and soluble proteins were extracted as previously described (Tanou et al., 2012 (link)). Protein concentration was determined following Bradford's method (Bradford, 1976 (link)), using BSA as standard. Proteins (50 μg) were separated by isoelectrofocusing on 3-10 NL IPG strips (11 cm; Biorad). The second dimension was carried out at 12.5% Tris-HCl polyacrylamide gels (Biorad). Three gels representing three biological replicates were run in parallel for each treatment and stained with silver nitrate. 2DE-gels were scanned with Bio-Rad GS-800 Calibrated Densitometer and analyzed with PDQuest Advanced 2-D Gel Analysis software (version 8.1, Bio-Rad) as previously described (Tanou et al., 2009 (link)). Statistical analysis was done by one-way analysis of variance (P < 0.05) and individual means were compared using Student's t-test (significance level 95%). The statistical significant differences were further combined by the quantitative two-fold change of spot volume (Supplementary Table S1).