S8), using Cell Quest software (BD PharMingen). For cytokine secretion analysis, cells were activated as described and the supernatants were assayed for IL-2 using ELISA (PeproTech, Rocky Hill, NJ, USA). All data were presented as average ± standard deviation (SD). Statistical significance was determined by Student’s t test; p < 0.05 was considered statistically significant. We gratefully acknowledge the support of the Society of Research Associates of the Lautenberg Center, the Concern Foundation of Los Angeles, and the Harold B. Abramson Chair in Immunology. This study
was supported by the US-Israel Binational Selleckchem Lapatinib Science Foundation (BSF), The Israel Sciences Foudation (ISF), The Israeli Ministry of Health, The Israel Cancer Research Foundation (ICRF), The Joint German-Israeli Research Program (DKFZ-MOST), and by The Joseph and Matilda Melnick Funds. We thank Prof. Muhlrad for the help with establishing the sedimentation assay, and Orly Perl for helping in performing the acceptor photobleaching FRET assay. The authors declare no financial or commercial Gefitinib supplier conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed
to the authors. Figure S1. The putative actin-binding motifs within the ζ-chain. (A) A schematic representation of the full-length ζ structure (upper panel) and the position of the two positively charged clusters (indicated by ellipses drawn
on the sequence, Urease lower panel) located within the ζintracytoplasmic domain. (B) The ζ positively charged clusters are evolutionarily conserved between species; the basic residues are labeled in bold letters. (C) The proximal RRR motif was mutated to GGG and the distal motif to QQQ. Figure S2. Mutations in the ζ positively charged motifs disrupt its dicf localization. A chart demonstrating ζ dscf (Ds) and dicf (Di) ratios as measured by densitometry analysis. The values are avarage of three independent transfections of the WT, Distal, Proximal or double mutated ζ chains into COS cells. The cells were lysed, dicf and dscf were separated and subjected to immunoblotting with anti-ζ antibodies as described in Fig. 1C. *P<0.003, **p<0.00003 Figure S3. The ζ positively charged motifs mediate its direct binding to actin. Mutations of the positively charged ζ motifs prevent its binding to F-actin. (A) WT and MUT IC ζ proteins were used in a dot blot assay for testing their capacity to bind F-actin. Associated proteins were detected using anti-ζ antibodies. (B) Biotinylated peptides containing the ζ positively charged (pepR) or mutated (pepQ) motif, were used in a dot blot assay. A representative experiment is shown out of at least three performed. Figure S4.