Fig. 1. Fgr binds FasL proline-rich domain (PRD) residues 41-60. (A) Amino acid sequences of the cytoplasmic tails of WT, -Pro, KKR and 3Y FasL. All constructs are tagged with GFP at the amino terminus and have wild-type transmembrane and extracellular domains at the carboxy-terminus. Amino acid numbering is shown with the PRD shaded and asterisks marking the positions of tyrosine and lysine residues. Amino acid substitutions are underlined for each construct and the deleted region in -Pro is shown. Please note that Figure 2a in a related paper (Blott et al. 2001) contains a typographical error, showing the sequence of C32 in FasL cytoplasmic tail as L. The correct sequence is shown here (A), and all constructs used in both studies corresponded to this sequence. (B) Western blots of pull downs using Fgr-SH3-GST incubated with cell lysates from untransfected RBL (-) or RBL expressing wild-type (WT) or mutant FasL GFP-tagged constructs lacking the PRD (-Pro), with K71, K72 and R73 all mutated to glutamic acid (KKR) or Y7, Y9, Y13 all mutated to alanine (3Y). Controls of the total lysate used in each pull down are shown. All blots were probed with anti-FasL, G247-4. Molecular mass markers are shown (kDa). (C) Different concentrations [shown above peaks (µM)] of monomeric Fgr-SH3 were passed over a random peptide (solid line), peptide 1-20 (light dotted line) and peptide 61-80 (bold dotted line) immobilised on a BIAcore^TM chip at 37°C. Protein injection points are indicated by horizontal bars. (D) Different concentrations [shown above peaks (µM)] of monomeric Fgr-SH3 were passed over peptides corresponding to amino acids 1-20 (dotted line) and 41-60 (bold line) immobilised on a BIAcore^TM chip at 37°C. Protein injection points are indicated by horizontal bars. (E) Specific equilibrium binding values were plotted (squares) and the K_D calculated by nonlinear curve fitting (line) and Scatchard analysis (see inset graph).