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Figure 4


Fig. 4. Identification of the EF1A1-binding motif in DLC1 SAM. (A,B) HEK293T cells were co-transfected with plasmids encoding (A) HA-EF1A1 and Flag-tagged SAM wild-type (WT), one double mutant K48A/R49A, one triple mutant F38G/L39G/F40G, or two quadruple mutants, K48A/R49A/R64A/R65A, F28G/F53G/L54G/A58G or a Flag-tagged control encoding an unrelated protein (Ctrl); or (B) HA-EF1A1 and Flag-tagged DLC1 SAM wild-type, one triple mutant, F38G/L39G/F40G, three single-point mutants, F38G, L39G, F40G or DLC2 SAM. Whole cell lysates (WCL) were used for immunoprecipitation (IP) with M2-anti-Flag beads. WCL and IP samples were immunoblotted with indicated antibodies. Asterisk indicates the light chain of Flag antibodies. The slower migrating bands for F38G/L39G/F40G, F38G, L39G and F40G mutants were due to their longer polypeptide chains (residues 1-94) compared with the wild type (residues 1-86). This slightly longer version was initially engineered to still encompass the full SAM (residues 11-76) but with eight amino acid residues flanking the C-terminus to help ensure proper folding of the SAM domain. These added residues essentially have no impact on the binding, as shown by F40G mutant and the WT. (C) Homology of DLC1 SAM with other SAM domains of known structures. Multiple sequence alignment was generated using the program CLUSTALW (http://www.ebi.ac.uk/clustalw). Residues totally conserved in all sequences are shaded black, those conserved in most of the sequences are in dark grey, whereas the significant but least conserved residues are in light grey. Species abbreviations: hs, Homo sapiens; mm, Mus musculus; sc, Saccharomyces cerevisiae. GenBank accession numbers: hsDLC1, 33188437; hsDLC2, 28976169; hsDLC3, 31543658; mmEPHA4, 30705030; hsEPHB2, 119615430; hsFLI1, 14603316; scSTE11, 609414; mmBAR, 21313130; scVTS1, 74583753; hsARAP2, 16118245; mmSAMSN1, 10800126. Asterisks indicate the corresponding residues of F38, L39 and F40 in DLC1 SAM.





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