08 44574 8.30 28% 100 Glycolytic Enzymes 756 gi|1125065 laminin-binding protein laminin receptor 3.05 14104 7.03 16% 98.157 Cytoskeletal/structural protein 830 gi|230867 Chain R, Twinning

In Crystals Of Human Skeletal Muscle GAPDH 4.16 35853 6.60 11% 100 Glycolytic Enzymes 888 gi|15277503 ACTB protein [Homo sapiens] b-actin 3.09 40194 5.55 17% 100 Cytoskeleton 952 gi|2780871 Chain B, Proteasome Activator Reg(Alpha) 3.71 16285 7.14 14% 99.989 Immunoproteasome assembly 976 gi|999892 Chain A, Crystal Structure Of Recombinant Human Triosephosphate Isomerase 4.12 26522 6.51 22% 99.594 Glycolytic Enzymes 1153 gi|6470150 BiP protein [Homo sapiens] 3.12 70888 5.23 41% 100 the chaperone family of protein 1158 buy LY2874455 gi|4503571 enolase 1 [Homo sapiens] 4.72 47139 7.01 41% 100 Glycolytic Enzymes * average ratio, B16M group/B16 group Figure 1 The images of representive 2D-DIGE and validation of vimentin. (A) A representative 2D-DIGE gel images. The extracted proteins were labeled with fluorescent dyes and separated by 2D-DIGE. B16M group was labled with cy3, B16 group was labled with cy5. (B) A representative two-dimensional gel

image. Differential expressed proteins that have been successfully identified by MALDI-TOF/MS (p ≤ 0.05, protein fold≥2) are circled and numbered. The spot numbers correspond to those proteins listed in Table 1. (C) The magnified protein spot images of vimentin in 2D gel showing the significant over-expression in B16M group compared with B16 group. (D) Western blotting shows changes in expression levels of vimentin in B16M group and B16M group; β-actin is used as the YH25448 ic50 internal loading control. (E) Histogram showing the relative expression levels of vimentin in eight pairs of B16M and B16 tissues, as determined by densitometric analysis (p = 0.021). Validation of vimentin expression by western blotting Western blotting was performed to verify the differential expression of vimentin in eight pairs of B16M group and B16 group. Equal expression of β-actin as internal standard was to identify the same protein loading. As shown in Figure 1D-E,

vimentin was significantly up-regulated in B16M group compared to B16 group (P < 0.05), which was consistent with the 2D-DIGE results. Expression of vimentin in melanoma patients We further detected the expression of vimentin using Non-specific serine/threonine protein kinase immunohistochemistry in 70 primary malignant melanoma patients to evaluate its clinicopathological significance. The differential expression of vimentin was shown in Figure 2A-B. Primary melanomas with overexpression of vimentin tends to have a more hematogenous metastasis incidence (P < 0.05). There is no statistical significance between overexpression of vimentin with age, gender, tumor location, TNM stage and lymph node metastasis (Table 1). Cox proportional hazards model analysis was performed and showed that the presence of TNM stage was a independent indicator of poor prognosis for melanoma patients (P = 0.004).