Pet28a

Codon optimized synthetic genes encoding His₆-FLAG-hIFNγ and His₆-FLAG-K88Q in pMA transfer vectors were synthesized by Life Technologies. After digestion with NcoI and XhoI the products were purified and ligated to the linearised vector pET28a (Qiagen) using Rapid DNA Ligation kit (Roche) for 15 min at 20°C. TOP10F' E. coli cells were transformed and the positive clones were selected by kanamycin resistance (70 µg/ml) followed by Colony PCR. Qiagen Qiacube Lab Robot was used for isolation of plasmid DNA which was further verified for sequence integrity by Eurofins Genomics, Germany.

The cDNA sequences encoding A. thaliana TOP1 (AT5G65620) and TOP2 (AT5G10540) were cloned into pET-28a (Agilent) in a translational frame with 6xHis N-terminal tags. Cysteine mutagenesis was performed by site-directed mutagenesis, using the wild-type cDNAs, gene-specific primers, and Phusion High-Fidelity DNA Polymerase (New England Biolabs). The PCR products were separated on agarose gels (0.8%), purified using the QIAquick gel extraction Kit (Qiagen), and cloned into pET-28a using NheI and SalI restriction enzymes (NEB) and T4 DNA ligase (NEB). The plasmids were used for the expression of TOPs in E. coli. Additional details are described in Supplemental Methods.

To raise antibodies against ZHP-4 and avoid cross-reactivity with other RING domain containing proteins, a fragment of 372 base pairs corresponding to the C-terminus of ZHP-4 was cloned into two bacterial expression vectors: pGEX-6p-2, containing the GST tag at the N-terminus (GE Healthcare) and pET28a (Qiagen), to generate an N-terminal 6xHis-fusion protein. Recombinant proteins were purified under native conditions using anti-GST beads (GE Healthcare) and Ni-NTA matrix (Qiagen) respectively following the manufacturer’s instructions. GST::ZHP-4 was used for antibody production in rat and 6xHis::ZHP-4 was used for sera purification (Medimab). ZHP-4 antibody was purified using activated supports according to the manufacturers’ protocols (Affi-Gel 10, BioRad).