Dpni enzyme

Residues were mutated to Alanine by PCR. NEBaseChanger web tool (http://nebasechanger.neb.com/) was used to design the primers for the mutagenesis and the primers are shown in Supplementary Table 3b. PCR reaction was performed in Q5 reaction buffer (New England Biolabs B9027S) supplemented with GC enhancer, with 18 ng plasmid DNA as the template, 200 μM each dNTP, 1U Q5 hot start high fidelity DNA polymerase (New England Biolabs M0493L), 500 nM each primer (Supplementary Table 3b), in a total volume of 40 μl. The reaction was performed at 98 °C for 30 followed by 25 cycles of 98 °C for 10 s, 58–69 °C (depend on the primers Ta) for 20 s, 72 °C for 150 s and final incubation of 72 °C for 2 minutes. PCR products were digested with DpnI restriction enzyme (New England BioLabs). The reaction was performed in cut smart buffer, with 10 μl of PCR products and 1 μl DpnI enzyme (New England Biolabs) in a total volume of 50 μl. The reaction was performed in 37 °C for 8 hours. After digestion, 10 μl of the reaction was transformed into DH5α competent cells as described above. Colonies were picked, grown in LB-Amp media overnight, and plasmids were purified with NucleoSpin Plasmid EasyPure kit according to manufacturer instructions (Macherey-Nagel) Plasmids were sent to sequencing to validate the mutation.

pVHL, SOCS2, Elongin C and Cul2 mutants were prepared by PCR-based method using the respective expression vectors encoding for the wild type proteins as template. The amplification of the expression vectors was performed using the KOD hot-start DNA polymerase (EMD Millipore), following the manufacturer guidelines and specific pairs of primers (Table S6) were used for the introduction of the desired mutation. PCR products were treated with DpnI enzyme (New England Biolabs) and transformed in DH5α E.coli cells. The mutations were confirmed by DNA sequencing.

To generate plasmids expressing the last 20 aa of ORF45 homologues fused to GST, we purchased synthesized oligonucleotides from Integrated DNA Technologies (sequences available upon request). For each homologue, the corresponding coding and anticoding strands with restriction digestion sites at the hanging ends were annealed and cloned into pGEX-5X (Pharmacia, now Cytiva, Marlborough, MA, USA). Mutant derivatives of each pGEX plasmid were generated as needed using the QuikChange protocol (Agilent Genomics, Santa Clara, CA, USA). Briefly, 125 ng each of sense and antisense primers encoding the desired mutation (primer sequences available upon request) were mixed with 10 ng of template, 1 × PFU enzyme buffer, 200 µM dNTP, and 1 µL PFU enzyme. Following PCR, 1 µL of DpnI enzyme (New England Biolabs, Ipswich, MA, USA) was added to each tube and the mix was then incubated at 37 °C for 1 h. Top10 E.coli cells were then transformed with 1 µL of the plasmid mixture and the resulting cells were grown overnight at 37 °C on LB-Amp plates. Positive clones were then isolated and the desired mutations were confirmed by sequencing.

hSTAT5B variants were generated using site-directed mutagenesis (80 (link)). Mutagenic PCR was performed using KOD Polymerase (Novagen). PCR products were subsequently digested with DpnI enzyme (New England BioLabs) to remove the methylated template according to the manufacturer’s protocol. E. coli was transformed with the digested reaction, and positive clones were selected by Sanger sequencing (81 (link)). Plasmid transfection was performed using Lipofectamine 2000 Reagent (Invitrogen, Thermo Fisher Scientific).
The cases of patients harboring the STAT5B^N642H mutation were assembled from previously published work (13 (link)–26 (link), 36 (link)).