Pmt bip v5 his a vector

The pIB vector (Invitrogen) was used for constitutive expression of the C. elegans DPY-19 protein (pIB-DPY-19) (Buettner et al., 2013 (link)). The pMT/BiP/V5-His A vector (Invitrogen) was used for expression of V5- and His-tagged C. elegans UNC-5 TSRs 1+2 and Drosophila Notch EGF repeats as well as His-tagged C. elegans UNC-5 TSR2 (corresponding protein sequences are depicted in Supplementary file 1).

To produce the near full-length of CLIPB9 and CLIPA14 transcripts for eukaryotic expression, cDNA was amplified by PCR using specific primers (Table S1). The PCR product was cloned into the pMT-BiP/V5-HisA vector (Invitrogen). Putative cleavage sites of proCLIPB9 and proCLIPA14 are IGMR¹³⁶ and LGFR¹⁶³. The four residues of the hypothetical cleavage site were replaced with the IEGR tetrapeptide recognized by Factor Xa (New England Biolabs) by the overlap extension PCR method, and the recombinant plasmids of proCLIPB9_Xa and proCLIPA14_Xa were obtained. The recombinant plasmid and pCoHygro hygromycin selection vector (Invitrogen) were co-transfected into Drosophila S2 cells cultured in SFX medium (HyClone) at 28 °C, and stable cell lines were selected. After induction with 500 μM copper sulfate, the supernatants were collected for recombinant protein purification. Then, protein was purified using Ni-NTA agarose. The concentration of the purified protein was estimated by bicinchoninic acid (BCA) assay and analyzed using SDS-PAGE followed by immunoblotting. As for recombinant PPO3, it was purified with Ni-NTA agarose as described previously (31 (link)). The purified products were stored at −80 °C until use.

The gene of AaHig was amplified and inserted into the pMT/BiP/V5-His A vector (Invitrogen), and then the recombinant plasmids were transfected into Drosophila S2 cells in combination with a Hygromycin selection vector pCoHygro for stable cell construction. The primers for PCR and gene cloning are shown in the S1 Table. The stable cell screening and purification were described in our previous study [2 (link),3 (link)]. Briefly, the transfected cells were selected using 300 μg/mL Hygromycin-B (Invitrogen, Cat. No# 10687–010) for 4 weeks. The resistant cells were grown in spinner flasks, switched to Express Five serum-free medium (GIBCO, Invitrogen, Cat. No# 10486025) for 3 days, and induced with copper sulfate at a final concentration of 500 μM for 4 days. The culture medium was collected for protein purification with a metal affinity resin (Clontech, Cat. No# PT1320-1). The protein was eluted with 150 mM imidazole, extensively dialyzed against PBS (pH 7.8), and concentrated via centrifugal filtration through a 5-kDa filter (Millipore Corp., Cat. No# pLCC07610). The protein concentration was measured using a Protein Assay Dye (Bio-Rad, Cat. No#500–0006) and a Nanodrop 2000c spectrophotometer (Thermo Scientific). The protein purity was checked with sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the specificity of purification was confirmed by western blotting.