cDNAs encoding the C-terminus of dtn-1 (a.a. 441-837), the C-terminus of dtn-2 (a.a. 434-818), and full-length pot-2 were cloned into the pET28c+ vector (Millipore). cDNA encoding the C-terminus of pot-1 (a.a. 100-300) was cloned into the pGEX-6P-1 vector (Addgene). The HIS- or GST-tagged recombinant proteins were expressed in BL21 (DE3) cells, solubilized in extraction buffer (600 mM NaCl and 50 mM Tris-HCl (pH 7.5)), and purified with Ni-nitrilotriacetic acid (QIAGEN) for the HIS tag or with glutathione agarose (Thermo Fisher Scientific) for the GST tag. The recombinant proteins were dialyzed into PBS and used to immunize rabbits. The polyclonal antibodies were affinity purified on antigen-coupled Sepharose beads (GE Healthcare).
pot-2