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Hierarchical Organization of Guidance Receptors: Silencing of Netrin Attraction by Slit Through a Robo/DCC Receptor Complex
Elke Stein and Marc Tessier-Lavigne

Supplementary Material

Construction of Recombinant Fusion Proteins and Baits
A detailed description of all constructs used in this study is available upon request. Briefly, constructs were made in one or more of the following vectors: the pCRII-TOPO cloning vector (Invitrogen), the yeast two-hybrid "bait" LexA fusion plasmid pBTM116, the "prey" VP16 plasmid pVP16, and the COS cell expression vectors pcDNA3 and pSEC-B (Invitrogen). DCC(HA), DCCec-TM(HA), myr-DCC, myrDCCΔP1, and rRobo1 were described previously [K. Hong et al., Cell97, 927 (1999); K. Brose et al., Cell96, 795 (1999)]. Various ectodomain or cytoplasmic domain fragments were derived by PCR from the rat DCC, rat Robo1, mouse Met, rat trkA, human EphB1 (accession numbers U68725, NM022188, AAA40579, AH000015, and AF03331, respectively). Overlap extension PCR was used to generate all chimeric receptors and deletion constructs by the method of S. N. Ho et al., Gene77, 51 (1989). SAM domain fusions are carboxyl-terminal in-frame fusions in which the EphB1 receptor donates AAs 880-984 to either Robo1 (AAs 1651) or a DCC construct omitting P3 (AAs 1420). The trkA-Robo chimeric constructs were generated with the rat trkA cDNA as a template [D. O. Clary, G. Weskamp, L. R. Austin, L. F. Reichardt, Mol. Biol. Cell5, 549 (1994)]. Rat trkA ectodomain sequences AAs 1-418 were fused in-frame with rRobo1 AAs 872-1651, in which Robo1 donates the transmembrane and cytoplasmic domain. The trkA-Robo1ΔCC1 deletion was generated with overlap extension PCR deleting AAs 1051-1100, using trkA-Robo as a template. All Met-chimeric receptors were composed of the Met ectodomain sequences AAs 1-930 using the mouse Met kinase [A. Iyer et al., Cell Growth Differ.1, 89 (1990)] as a template. For Met-DCC, DCC donates AAs 1095-1444, which encompass the transmembrane and cytoplasmic domains of DCC. Met-DCCΔP1 was generated omitting DCC AAs 1149-1166. Met-DCCΔP3 constructs contained DCC AAs 1095-1420, followed by the HA tag directly or the SAM domain as described above. A myristoylated Robo1 fusion construct was generated by PCR, placing a Hind III site upstream of the src-myr sequence (MGSSKSKPKDPSQRRRSLE) [B. Guy et al., Nature330, 266 (1987)] in-frame with the Robo cytoplasmic domain (AAs 907-1651) and cloned into pSEC-B. The GFP cDNA containing a mutation at S65T to increase the intensity of the green fluorescence was described previously [X.-H. Wang, M.-M. Poo, Neuron19, 825 (1997)]. All PCR fragments and in-frame cloning sites generated were confirmed by sequence analysis. The integrity of all constructs used in the axon-turning assay was assessed by in vitro transcription and translation with the Transcend Non-Radioactive Detection System (Promega Co., Madison, WI).