Figure 3

Beta-aggregation prediction of the SH2D2A SH2 domain. A. Amino acid positions of the indicated SH2 domains (X-axis) plotted against the corresponding beta-aggregating value (Y-axis). B . Beta-aggregation values of amino acids 122–135 in wild type (WT) and in silico mutated SH2D2A encoding TSAd. C. Ribbon diagrams and space filling models of the Lck SH2 domain (PDB ID: 1BHH) and the ALX SH2 domain (PDB ID: 2CS0) showing the location of Ser-Phe-Ser (Lck) and Gly-Tyr-Thr (ALX) corresponding to the TSAd TFV sequence. Models show that the side chains of Ser 161 and Ser 163 in Lck SH2 and Gly65, Tyr66 and Thr67 of ALX SH2 are solvent accessible. D.-F. WT and mutated SH2 domains expressed in E.coli at 15°C. D. Equal amount (5 μl) of soluble fraction from a 100 ml bacterial culture was separated by SDS-page and immunoblotted with anti-GST antibodies for quantification of soluble recombinant protein. GST = 1 equals 0,11 μg purified GST protein. One representative experiment out of three is shown. E. Quantitation of soluble GST-SH2 proteins by Image J analysis based on D. ND; not detected. F. Soluble protein captured on glutathione beads were processed as in Fig. 2B. G. Solubility of WT and mutated TSAd SH2 (5-AS, TSAd-90-188-PAAS) (GYT) and ALX SH2 (TFV) expressed in E.coli. Uninduced E.coli (U), soluble (S) and pellet (P) fractions were processed as in Fig. 2B. Amount of soluble and insoluble SH2 domain visualized by anti-GST immunoblotting (upper panel). Total protein content in samples was visualized by Coomassie Brilliant Blue Staining (lower panel). H. CD3 stimulated Jurkat cell lysates (S) and proteins pulled down with the indicated SH2 domains were separated by SDS-page and immunoblotted. I. Peptide array with VEGFR-2 and VCP phosphotyrosine peptides probed with the indicated SH2 domains. Bound SH2 domains detected using anti-GST antibody.