pHT58

This plasmid was designed to express a soluble UNC-40 extracellular domain fused to human IgG Fc (UNC-40-Fc) in mammalian cells under the control of CBA promoter. The coding sequence of UNC-40 ectodomain (residues #26 to#1085) was inserted in the pHT48 vector between NheI and BamHI sites using isothermal assembly (Gibson, 2011). This plasmid was used to express secreted UNC-40-Fc in HEK293cells for the co-immunoprecipitation of MADD-4A or B isoforms with UNC-40.

pHT28

Three Myc tag repeats were inserted after the signal peptide of UNC-40 (after residue #25) using fusion PCR. The sp::3xMyc-unc-40 and unc-54 3'UTR fragments amplified from pHT25 were fused by fusion PCR strategy. Finally, the entire sp::3xMyc-unc-40::unc-54 3'UTR fragment was inserted into the pPD115.62 vector between KpnI and Bps120I sites.

pHT25

The unc-40-GFP cDNA derived from DACR104 (a kind gift from D.A. Coln-Ramos) was inserted into pPD115.62 (L3570, A. Fire kit) using blunted EcoRI and Bps120I sites, the GFP sequence was fused at the Cterminal end of UNC-40.

pVB518OB

Clone = pVB518OB. | Using an oligo dT primed first strand cDNA reaction from total C.elegans RNA, the full length tomm-40 cDNA was amplified using primers with flanking AgeI sites. The amplified cDNA, which was designed to lack the stop codon, was inserted between the tomm-40 promoter and the gfp coding region in pVB488OB at the AgeI site to yield pVB518OB which contains a full length tomm-40::gfp fusion construct.

pVB559MA

Clone = pVB559MA. | tomm-40 genomic DNA was amplified using primers with flanking NheI sites corresponding to the start and to the stop of the gene, but lacking the start and stop codons. This fragment was inserted as an NheI/NheI fragment into the pVB299GK plasmid to generate Posm-6::tomm-40, where tomm-40 was inserted in a 5'to 3' orientation. The orientation was confirmed by sequencing.

pVB560MA

Clone = pVB560MA. | tomm-40 genomic DNA was amplified using primers with flanking NheI sites corresponding to the start and to the stop of the gene, but lacking the start and stop codons. This fragment was inserted as an NheI/NheI fragment into the pVB299GK plasmid to generate Posm-6::tomm-40, where tomm-40 was inserted in a 3'to 5' orientation. The orientation was confirmed by sequencing.

pBP39

This plasmid was used to express MADD-4A-MYC in HEK293 cells for co-immunoprecipitation with UNC-40-Fc.

pBP41

This plasmid was used to express MADD-4B-MYC in HEK293 cells for co-immunoprecipitation with UNC-40-Fc.

pSL779

N2 animals were injected with pSL779 at 40 ng/L + 100 ng/L of odr1::rfp to make strain UD522 [ycEx249].

pHT31

A myristoylation signal (MGSSKS) was added between the signal peptide (residue #1 to #25) and the intracellular domain of UNC-40 (residues #1108 to #1415) using fusion PCR. The pHT25 plasmid, with the exception of the extracellular domain and the transmembrane region of UNC-40 (residues #26 to #1107), was amplified by using reverse PCR. The coding sequence of the myristoylation signal was added in the forward primer and NheI sites were added in both ripmers. The PCR fragment was religated after digestion by NheI.