1 Introduction Negrutskii & El'skaya, 1998 Petrushenko et al., 1997 Petrushenko, Shalak, Budkevich, Negruskii, & El'skaya, 2002 Lamberti et al., 2004 Chang et al., 2002; Kim et al., 1999 Maruyama, Nara, Yoshikawa, & Suzuki, 2007 Lau, Castelli, Lin, and Macaulay (2006) Kahns et al., 1998 Amiri et al., 2007; Anand et al., 2002 In vivo in vitro 2 Materials and methods 2.1 Cell lines and plasmid constructs HEK293 cells were purchased from the American Type Culture Collection (Manassas, VA) and grown according to their instructions. To produce eEF1A1 and eEF1A2 stable cell lines pcDNA 3.1 eEF1A1 and eEF1A2 with the C-terminal His-tags were linearized by MunI and transfected in HEK293 cells using ExGene500 (Fermentas) according to manufacturer recommendations. G418 was used to select stable cell lines. 2.2 Bioinformatics Obenauer, Cantley, & Yaffe, 2003 2.3 Purification of the eEF1A1 and eEF1A2 isoforms Budkevich et al., 2002 3 Carvalho, Carvalho, & Merrick, 1984 2.4 Pull down assay Gout et al., 1993 2.5 Phosphatase treatment 2 g 2.6 Immunoprecipitation g 2.7 Immunoblot analysis Proteins were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and incubated for 1 h with blocking solution (2% BSA, Tris-buffered saline, 0.1% Tween-40). After blocking the membranes were probed 2 h at 4 °C with anti-GST, anti-eEF1A (Upstate Biotechnology), anti-His tag (Cell Signalling), anti-Shp2 (Cell Signalling) or anti-pTyr 4G10 (Upstate Biotechnology) antibodies. After extensive washing with the solution of Tris-buffered saline and 0.1% Tween-40, the membranes were incubated with secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. The antigen–antibody complexes were detected using the ECL system (Millipore). When immunoblots had to be re-probed, the membranes were initially stripped and re-blocked prior to incubation with another type of primary antibody. 3 Results 3.1 Existence of SH2/SH3 domain binding sites in the eEF1A1 and eEF1A2 molecules are predicted by bioinformatic analysis Obenauer et al., 2003 Fig. 1 3.2 eEF1A1 and eEF1A2 interact in vitro with SH2/SH3 domains of different proteins 2 Fig. 2 Kim et al., 1999 Chang et al., 2002 Frackelton et al., 2006; Tomlinson et al., 2005 Kahns et al., 1998 Feller, 2001 Gross & Kinzy, 2005 Shiina, Gotoh, Kubomura, Iwamatsu, & Nishida, 1994 Alper & Bowden, 2005 Amiri et al., 2007; Lamberti et al., 2004; Lee, 2003 3.3 eEF1A1 and eEF1A2 are in complex with Shp2 in vivo in vitro in vivo Fig. 3 in vitro Fig. 3 Fig. 3 Ann et al., 1991 in vivo 3.4 eEF1A2 and Shp2 interaction is sensitive to dephosphorylation Fig. 3 3.5 eEF1A1 and eEF1A2 are phosphorylated at Tyr in vivo in vitro in vivo Fig. 4 Fig. 4 4 Discussion Gross & Kinzy, 2005 Lamberti et al., 2004; Lee, 2003 Ahmed, Forsberg, & Bergsten, 2005 In vitro Lee, 2003 Chang & Wang, 2007 Schlessinger & Lemmon, 2003 Li, 2005 Fig. 2 Fig. 2 Mohi & Neel, 2007 Burks & Agazie, 2006 Harada, Katoh, & Negishi, 2005 Murray, Edmonds, Liu, & Condeelis, 1996 Nandan, Yi, Lopez, Lai, & Reiner, 2002 Yarwood, Bouyoucef-Cherchalli, Cullen, & Kupzig, 2006 Mansilla et al., 2002 Fig. 4 Rush et al., 2005 in vivo Anand et al., 2002