Differences between control and treated cells were assessed using one-way ANOVA and a significance level of P < 0.05 was required. Results Comparative proteomics EPZ015666 datasheet analysis The silver-stained 2D-PAGE profile of the PcDNA3.1(IGFBP7)-RKO
transfectants and the PcDNA3.1-RKO -transfectants revealed approximate 1100 staining spots (1171 ± 109 vs 1120 ± 80), respectively. Using a 3-fold criterion for selecting, 12 protein spots were visually detected as significantly differentially expressed between the two groups. The representative images, emphasizing the location of the 12 protein spots on the gel were shown in SB525334 nmr Figure 1. Interestingly, of the 12 spots, only one spot was upregulated (spot 12) and the other 11 spots were downregulated in the cell lysates of NVP-HSP990 datasheet PcDNA3.1(IGFBP7)-RKO transfectants. Figure 1 2D electrophoresis profiles of PcDNA3.1( IGFBP7 )-RKO-transfectants and PcDNA3.1-RKO transfectants. A. 2D electrophoresis profiles of silver staining proteins of PcDNA3.1(IGFBP7)-RKO transfectants (BP7-RKO) and PcDNA3.1-RKO transfectants (control). 0.75 milligrams of protein were loaded onto linear IPG strips (pH 5-8) and isoelectric focusing was performed at 35 kV-h. The second dimensional run was performed on 12.5% Tris-glycine-PAGE gels
and the gels were stained with silver for image analysis. Protein spot discrepancies were arrowed and marked with number. B. Close-up image of differential expression
of protein spots. MS based identification The above 12 differentially expressed protein spots were selected and submitted to MS based identification. As a result, 10 spots were identified by MALDI-TOF MS, representing 6 unique proteins, including albumin (ALB), HSP60, Actin cytoplasmic 1 or 2, pyruvate kinase muscle 2(PKM2), beta subunit of phenylalanyl-tRNA synthetase(FARSB) and hypothetical protein (Table 1). Two protein spots (spot 11 and spot 12) could not be identified, possibly due to the lower amount of protein as revealed by a retrospective analysis of the spot volumes. Of the 6 proteins identified above, all were found decreased in PcDNA3.1(IGFBP7)-RKO transfectants. Table 1 Characteristics of proteins identified from PcDNA3.1(IGFBP7)-transfected RKO cells and controls Spot Protein description Sequence coverage(%)* Swissprot ID Theoretical Mr/Pi** 1 Serum Idoxuridine albumin 5.74% P02768 69367/6.42 2 Serum albumin 7.97% P02768 69367/6.42 3 Serum albumin 6.86% P02768 69367/6.42 4 pyruvate kinase, muscle 22.45% Q9UK31 6002/7.58 5 Phenylalanyl-tRNA synthetase beta chain 12.56% Q9NSD9 66130/6.39 6 Actin, cytoplasmic 1 or 2 33.33% P63261 41793/5.31 7 Actin, cytoplasmic 1 or 2 23.20% P63261 41793/5.31 8 60 kDa heat shock protein, mitochondrial precursor 2.96% P10809 61055/5.7 9 60 kDa heat shock protein, mitochondrial precursor 28.52% P10809 61055/5.7 10 Hypothetical protein 21.49% P04406 36053.05/8.