Inase 6 14* 0.04 1.40 Flotillin-1 Alpha-enolase 16* 0.04 -1.32 Cdc42-i…

Inase 6 14* 0.04 1.40 Flotillin-1 Alpha-enolase 16* 0.04 -1.32 Cdc42-interacting protein 4 78 kDa glucose-regulated protein (GRP78) Collagen type IV alpha-3 binding protein NADH-ubiquinone oxireductase 75 kDa subunit, mitochondrial Stress-70 protein, mitochondrial 22* 0.04 -1.32 cDNA FLJ5299 Keratin, type II cytoskeletal 8 Thioredoxin domain-containing protein 5 Actin, cytoplasmc 1 26S protease regulatory Celecoxib subunit 6B Uncharacterised protein KIAA0174 (ISTI homolog) Eukaryotic initiation factor 4A-1 Hsc70-interacting protein Heterogenous nuclear ribonucleoprotein FaACC. No. (Swissprot) P07355 P60891 P45880 P52564 O75955 P06733 Q15462 P11021 Q9Y5P4 P28331 P38646 A8K781 P05787 Q8NBS9 P60709 P43686 P53990 P60842 P50502 PGene ANXA2 PRPS1 VDAC2 MAP2K6 FLOT1 ENO1 TRIP10 HSPA5 COL4A3BP NDUFS1 HSPA9 cDNA FLJ5299 KRT8 TXNDC5 ACTB PSMC4 KIAA0174 EIF4A1 ST13 HNRNPFSequence coverage ( ) 38.6 15.1 17.70 11.4 62.3 6.68 50.6 47.6 20.7 13.2 11.5 47.0 46.8 27.3 26.4 22.7 14.6 16.0 12.5 19.No. peptides identified 13 4 5 3 23 2 22 32 9 8 7 17 25 11 9 9 5 5 4Mr/ pI 38/ 7.56 35/ 6.56 32/ 7.5 37/ 7.01 47/ 7.08 47/ 6.99 69/ 5.55 72/ 5.01 71/ 5.29 79/ 5.42 74/ 5.44 47/ 5.11 48/ 5.34 48/ 5.37 42/ 5.29 47/ 5.09 40/ 5.22 46/ 5.32 41/ 5.18 46/ 5.Asterisk (*) denotes spots identified as containing a mixture of 2 or more proteins, or where protein isoforms cannot be distinguished based on peptides identified. P-value and fold-change obtained based on normalised spot volume between control and IL11 treated group.Validation of membrane protein abundance changes between IL11 treated and control treated hEEC cellstissues from women with normal fertility throughout the menstrual cycle (n = 6-8/cycle).IL11 increased Annexin A2 and Flotillin-1 proteins in hEEC membranesProtein changes observed between IL11 and control treated cells were validated by Western blotting and immunocytochemistry. Proteins were validated by western blot in two hEEC cell lines: ECC-1 and Ishikawa cells whilst, immunocytochemistry was carried out in ECC-1 cells and primary hEEC. Endometrial biopsies were also used to assess the cellular localisation and relative level of protein production in vivo. Immunohistochemical validation was performed using endometrialDIGE analysis identified that annexin A2 (ANXA2) in protein spot 9 (Table 1) was significantly increased following IL11 treatment. A mixture of four proteins were identified in spot 9 and ANXA2 was chosen due to the highest number of peptides present in the analyses and our interest in its biological function. Western blot analysis revealed that ANXA2 was present as a single bandYap et al. Reproductive Biology and Endocrinology 2011, 9:73 http://www.rbej.com/content/9/1/Page 8 ofof molecular weight 37 kDa (Figure 3A-C) and was significantly increased in the membrane protein fraction in IL11 treated compared to control in both the primary hEEC (Figure 3A, right panel) and hEEC-derived cell lines; ECC-1 (Figure 3B, left panel) and Ishikawa (Figure 3C, left panel) but no differences were observed in the total protein fractions of IL11 treated compared to control treated primary hEEC (Figure 3A; left panel). However, there appear to be a difference in abundance in IL11 treated membrane fraction but no difference in the cytosol fraction in both the control or IL11 treated (Figure 3A; right panel). In addition, Western blot showed that ANXA2 abundance was upregulated 2-fold and 2.5fold in IL11 treated compared to control in the cell membrane fraction.