Decyder software

The total albumin and globulin of flag leaves and developing grains were extracted according to Zhang et al. (2014) (link) with slight modifications. Mixing pairs of Cy3- and Cy5-labeled protein samples with a Cy2-labeled internal standard were subjected to two-dimensional difference gel electrophoresis (2D-DIGE). The DIGE images were analyzed using DeCyder software (ver. 6.5; Amersham, Little Chalfont, United Kingdom). 2D-DIGE analysis was based on Rollins et al. (2013) (link) and Cao et al. (2016) (link). Details on the 2D-DIGE experiments for differentially accumulated protein (DAP) identification and expression analysis are listed in Supplementary Table S1. Protein labeling, 2D-DIGE, imaging, and image analysis were performed according to Bian et al. (2017) (link) and Li et al. (2017) (link), with minor modifications. Only those with significant and biological reproducible changes (abundance variation at least two-fold, Student’s t-test, p < 0.05) were considered to be DAP spots. Three biological replicates were used for all samples.

Gels were run in triplicate, scanned, and analyzed as described previously [44 (link)]. To summarize, scanning was performed using a Typhoon Trioscanner (Amersham BioSciences) following the manufacturer’s protocol. Scanned images were processed using Image Quant software (Amersham BioSciences, v.5.0). Protein abundance was assessed by differential in-gel analysis (DIA). The quantitative analysis of protein spots was performed using DeCyder software (Amersham Biosciences, v.6.5). Quantitative comparisons were calculated between samples run at the same time and pair-wise volume ratios were calculated for each protein spot and used to determine relative fold changes ratios. Fold changes were log₂ transformed. Student’s t-test was performed using the log2 normalized average spot volume ratios for all spots detected from the three replicate experiments. Only statistically significant results (P<0.05), and differentially abundant proteins with a ratio > 2-fold (log2 of 1) difference in one condition (increase or decrease in abundance), were chosen for mass spectrometry.

2D-DIGE analysis was performed in nucleolar extracts from control and CAPN2 knocked-down MDA-MB-231 cells (n = 4). Nucleolar samples from scRNA and siCAPN2-transfected cells were alternatively labelled with Cy-3 or Cy-5 to decrease inter-gel variability. Recombined samples from silenced and control cells were labelled with Cy-2 for normalization and separated on 24 cm Immobiline DryStrips (pH 3–11) and 12.5%-acrylamide SDS-PAGE gels. Gels were scanned with Typhoon 9400 Variable Mode Imager and image analysis performed using DeCyder software (v7.0, Amersham Biosciences, GE Healthcare) and SameSpots v5.0.1.0. Significance of statistically different proteins was calculated using Student’s t-test and accepted when the value was p ≤ 0.05. Spots from 11 differentially represented proteins were excised and identified by LC–MS/MS at the proteomics facility (SCSIE) at the University of Valencia.