Pdest15

DNA fragments coding for CIT-K fragments (kinase, CC1, CC2, C1 + PH and CNH), Aurora B, Borealin, INCENP_1-261 and INCENP_525-918 were generated by PCR and cloned into pDEST15 (Invitrogen) to express N-terminal GST-tagged polypeptides in Escherichia coli. The GST-tagged products were then purified using Glutathione Sepharose 4B according to manufacturer's instruction (GE Healthcare). [³⁵S] Methionine-labelled Aurora B, Borealin, INCENP (all three fragments) and CIT-K fragments (kinase, CC2 and CNH) were prepared from corresponding PCR products amplified using primers harbouring a T7 promoter and then in vitro transcribed and translated (IVTT) using the TnT T7 Quick Coupled Transcription/Translation System (Promega) in the presence of [³⁵S] methionine (PerkinElmer). The binding reaction contained 150 mM NaCl and subsequent washes varied from 150 mM to 1 M NaCl. GST pull-down assays were carried out as described [12 (link)].

All expression vectors were generated using the Gateway system (Invitrogen). For MBP and GST tagging, we used pKM596 (Addgene) and pDEST15 (Invitrogen), respectively, as destination vectors. Flag and Myc tagging of Ana2 and Sas6 proteins for the co-immunoprecipitation experiments was achieved using the pAFW, and pAWM destination vectors from the Drosophila Gateway Vector Collection (https://emb.carnegiescience.edu/labs/murphy/Gateway%20vectors.html). The QuickChange Mutagenesis Kit (Agilent) was used to introduce all deletions and amino acid substitution mutations. The constructs were verified by DNA sequencing.

The full length gene of ct712 and the region of ct619 truncated of the first 81 and last 625 codons were amplified by PCR, and were cloned using the Gateway system into the pDEST15 (Invitrogen) destination vector, providing a GST tag at the N-terminus. Expression of the recombinant proteins was made after transformation of BL21 E. coli strain. BL21 bacteria transformed with the GST-Δ81CT619Δ625 construct were cultured in LB media supplemented with ampicillin at 37°C until the optical density at 600 nm reached 0.6 before addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) for expression induction. The GST-CT712 construct was obtained from cultures in micro-fermentors to overcome low yields. Protein purification was performed on column using glutathione-sepharose beads (GE Healthcare) following the manufacturer indications. Purified proteins were used to immunize New Zealand White rabbits for production of polyclonal antisera (AgroBio, La Ferté Saint-Aubin, France). To test these antibodies by western blot, cell lysates or purified EBs were lysed in 8 M urea, 1% SDS (v/v), 150 mM NaCl, 30 mM Tris pH 8.0 and the proteins were analyzed by SDS-PAGE as described below.

The full-length cDNAs of CaKAN3, CaKAN4 and CaHSFA1,CaHSF8 were amplified by PCR using specific primer pairs (Supplementary Table 1) and then cloned into the entry vector pDONR207 by BP reaction using a Gateway cloning system (Invitrogen, 11789020). To construct the CaKAN3 or CaHSF8 destination vectors for the overexpression assay or prokaryotic expression, the full-length CaKAN3 and CaHSF8 genes were cloned into pEarleyGate plasmid vectors: pEarlyGate101, pEarlyGate103, pEarlyGate201, pEarlyGate202^{99 (link)}; pSPYCE, pSPYNE^{100 (link)}; pPGCL, pPGNL^{101 (link)}, pDEST-17 (Invitrogen, 11803012) or pDEST-15 (Invitrogen, 11802014) by LR reaction.
To construct the vectors for the VIGS assay, the specific gene fragments of CaKAN3, CaHSF8 or NLRs in their 3’UTRs to avoid the possible silencing of their homologous genes were amplified by PCR using specific primer pairs (Supplementary Table 1), and each fragment was cloned into the entry vector pDONR207 by BP reaction individually and then into the destination vector pPYL279 by LR reaction using the Gateway cloning system (Invitrogen, 11791020).

A specified region in a gene’s CDS or 3′ or 5′ UTR was amplified with PCR using specific primer pairs (Table S1) to create a vector for gene silencing; similarly, a full-length ORF of a Ca16R was amplified using PCR employing appropriate primer pairs (Table S1) to create a vector for overexpression. The PCR product was then cloned using a Gateway cloning system (Invitrogen, 11789020) into the entry vector pDONR207 with BP reaction. To construct a vector for overexpression, the subcellular location assay, and the BiFC assay, the ORF was further cloned from the entry vector to the pEarleyGate plasmid vectors pEarlyGate103, pEarlyGate201 [61 (link)]; pSPYCE, pSPYNE [62 (link)]; pDEST-17 (Invitrogen, 11803012) or pDEST-15 (Invitrogen, 11802014) using LR reaction. To construct a vector for the gene-silencing assay, the specific gene fragment in CDS or 3′ or 5′UTR of a given gene was cloned into the destination vector pPYL279 using LR reaction.

3′-UTR regions of LDLR mRNA were cloned into pDEST12.2 (Invitrogen), which contains a 5′-RFP tag. 3′-UTR regions of β-actin mRNA were cloned into pDEST12.2 (Invitrogen), which contains a 5′-LUC2 tag. Human ZFP36, ZFP36L1 and ZFP36L2 open reading frames were cloned into pDEST12.2 (Invitrogen), which contains a 5′-MYC tag or a 5′-Flag tag, or into pDEST15 (Invitrogen).