Lr clonase reaction

The ARF2A ORF was cloned into PacI/AscI sites in pENTR/D-TOPO, and was introduced into pDEST32 vector using an LR clonase reaction (Invitrogen, USA; pDEST32-ARF2). These vectors were transformed into PJ69-4α yeast strain [72 (link)] by standard LiAc transformation (Yeast protocols handbook, Invitrogen, USA, July 2009) and used as the bait. The cDNA library used as the prey represented genes expressed in tomato fruit from breaker ripening stage and onwards. The library was cloned in pDEST22 vectors which together with the yeast strains were obtained from Prof. Richard Immink (Plant Research International B.V., Wageningen, The Netherlands). The interaction screenings were carried out using standard manufacturer’s protocol (ProQuest Two-Hybrid System with Gateway Technology, Invitrogen, USA), by two step transformation of the bait and prey vectors. Positive interactions were determined as positive growth on SD medium lacking leucine, tryptophan, histidine and adenine. Positive interactions were verified in a one-on-one yeast-two-hybrid. The interactor was identified through sequencing of the prey vector.

Expression constructs were generated with a N‐terminal Strep‐HA‐tagged bait proteins and entry clones of a Gateway compatible human clone collection (ORFeome v5.1 and v8.1). The integration of the entry clones into the Gateway destination vectors (pcDNA5/FRT/TO/SH/GW; Glatter et al, 2009 (link)) was performed with an enzymatic LR clonase reaction (Invitrogen). YAP1 mutant sequences were generated by gene synthesis in pDONR223 (Biocat GMBH) and subsequently cloned into pcDNA5/FRT/TO/SH/GW as described above. The sequences of YAP1 mutants oligonucleotides are reported in the Reagents and Tools table.

UBASH3B and LUBAC expression plasmids were generated by enzymatic LR clonase reaction (Invitrogen) of a UBASH3B pDONR vector (Orfeome v5.1) and a pTO-SH entry vector, encoding a C- or N-terminal Strep-HA tag. The UBASH3B expression vector and pOG44 vector (Invitrogen) were cotransfected in HEK Flp-In 293 T-Rex cells (Invitrogen) using X-tremeGENE transfection reagent (Invitrogen). Two days after transfection, cells that had undergone recombination were selected using DMEM supplemented with 100 μg/mL of hygromycin (Invitrogen) and 19 μg/mL of blasticidin (Huberlab) for 2 to 3 wk.

The vector of pENTR-D-Topo-β1 was previously established in our laboratory47 (link). We then used a Gateway^TM cloning System kit (Invitrogen) for getting the expression vectors. Briefly, a LR clonase reaction (Invitrogen) was used to transfer the cDNAs of β1 from the entry vectors into CSII-EF-Rfa. The CSII-EF-β1 was cotransfected with pCAG-HIVgp and pCMV-VSV-G-RSV-Rev into 293T cells. After infection for 48 h, the virus media was collected. The KO cells were infected with the resultant virus for 72 h, and the β1 positive cells were selected using the FACSAria II (BD Biosciences) twice. The stable cell line was used in subsequent studies.

For the generation of expression constructs encoding a N- or C-terminal twin-Strep and hemagglutinin (SH)-tag (pTO-SH) for affinity purification experiments or a N- or C-terminal FLAG-BirA*-tag^{51 (link)} for BioID experiments entry clones of a Gateway compatible clone collection (ORFeome v5.1) were used. The integration of the entry clones into the Gateway destination vectors was performed with an enzymatic LR clonase reaction (Invitrogen). Site-directed mutagenesis of Dyrk2 constructs was performed using Pfu Ultra High Fidelity DNA-polymerase according to the manufacturer’s instructions (Agilent Technologies). For the generation of the MultiBac vector expressing FLAG-tagged Dyrk2 (pFBDM-FLAG-Dyrk2) in SF9 cells, the coding sequence of Dyrk2 was amplified by PCR using oligonucleotides encoding the FLAG-tag and subcloned into pDNOR221 with BP clonase reaction (Invitrogen) (Supplementary Table 5). The DNA region encoding FLAG-Dyrk2 was inserted into pFBDM using NotI and HindIII restriction sites. The bacmid for the transfection of SF9 cells was generated by heat shock transformation of DH10Bac E. coli cells (#10361012, Invitrogen) with 2 µg pFBDM-FLAG-Dyrk2 and recovery at 37 °C for 8 h. Afterwards, cells were spread on agar plates containing Ampicillin, Kanamycin, Tetracycline, and Gentamycin. Grown colonies were used for the preparation of the bacmid DNA.