Specifications table br Value of the data

Specifications table
Value of the data
Data, experimental design, materials and methods Using the 2D-DIGE approach we have obtained relevant information on the changes in abundance of most nucleoside analog implicated in major canonical metabolic pathways [1]. Supplementary Table 1 lists all the identified proteins whose abundance differs by at least 1.5-fold (p<0.05, student t-test) between normal astrocytes (NA) and transformed astrocytes (TA). Our results suggest that transformation causes major losses of astrocyte-specific proteins and functions and the acquisition of metabolic adaptations that favor intermediate metabolites production for increased macromolecule biosynthesis. We also observe a loss of some enzymes implicated in the oxidative stress defense. This is illustrated in Supplementary materials showing zoomed-in 2D gels regions where the spots containing the proteins/enzymes that underwent these changes (e.g. Pyruvate kinase, Lactate dehydrogenase, Glutamate dehydrogenase, Glutamine synthetase, Transaldolase, Peroxiredoxin 1 and 6, Glutathione S-transferase Mu1…) are visible, together with a graphical representation of the quantitative data.
Specifications table
Value of the data
Data Total proteins in Brassica napus leaves were extracted from the chlorophyll-deficient mutant cde1 and the corresponding wild-type (WT) plants in three independent biological experiments using the iTRAQ approach. The distribution of lengths and numbers of peptides, mass and sequence coverage of proteins were shown in Fig. 1. Of the 5069 proteins identified in this study, over 60% of the proteins included at least two peptides and the sequence coverages of over 45% of the proteins were higher than 10%. Proteins with molecular masses too small or too large are difficult to be identified using traditional 2D gel technique. 145 low molecular weight proteins (Mr<10kDa) and 521 high molecular weight proteins (Mr>100kDa) were identified using iTRAQ strategy. To demonstrate the repeatability of the replicates from the wild-type and cde1 mutant, the protein abundances between various biological replicates were compared (Fig. 2). The results indicated that about 80% of the proteins showed less than 1.5-fold change between biological replicates. Changes in the protein profile in response to cde1 mutation were analyzed and 443 proteins showed a significant difference (p-value<0.05) with the false discovery rate (FDR) less than 5%. Among these proteins, 35, 116, and 228 proteins reproducibly decreased to less than 0.50-, 0.67-, and 0.83-fold, respectively (Supplementary Table 1). On the other hand, 33, 96, and 215 proteins increased by more than 2.0-, 1.5-, and 1.2-fold, respectively (Supplementary Table 2). Functional analyses of these differentially accumulated proteins were performed using Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The results showed that the down accumulated proteins included important enzymes in porphyrin and chlorophyll metabolism, carbon fixation and secondary metabolites biosynthesis. The abundance of photosynthetic proteins and proteins related to redox homeostasis was significantly reduced in the cde1 mutant. Proteins that involved in spliceosome, mRNA surveillance, RNA degradation and protein modification were up accumulated in cde1, suggesting important roles of posttranscriptional RNA processing and posttranslational protein processing in the regulation of chlorophyll biosynthesis.
Experimental design, materials and methods Fig. 3 shows the experimental design used to gather the data presented here and in [1].
Specifications table
Value of the data
Experimental design, materials and methods 100ml of SKY4364 [3] were grown to an OD600 of 0.5 in YPD media. Cells were split into two flasks, one untreated and one which was subjected to 0.02% MMS for 3h. Cells were harvested and HIS6-tagged Ssa1 along with the associated interactome was isolated as follows: Protein was extracted via bead beating in 500µl Binding/Wash Buffer (50mM Na-phosphate pH 8.0, 300mM NaCl, 0.01% Tween-20). 200µg of protein extract was incubated with 50µl His-Tag Dynabeads (Invitrogen) at 4°C for 15min. Dynabeads were collected by magnet then washed 5 times with 500µl Binding/Wash buffer. After final wash, buffer was aspirated and beads were incubated with 100µl Elution buffer (300mM imidazole, 50mM Na-phosphate pH 8.0, 300mM NaCl, 0.01% Tween-20) for 20min, then beads were collected via magnet. The supernatant containing purified HIS6-Ssa1 was transferred to a fresh tube, 25µl of 5× SDS-PAGE sample buffer was added and the sample was denatured by boiling for 5min at 95°C. 10µl of sample was analyzed by SDS-PAGE. To isolate HIS6-tagged Hsp82, SKY4635 expressing HIS6-Hsp82 as the sole Hsp90 isoform in the cell [4] were grown and processed identically to the SKY4364 cells as above.