Ptag gfp2 c vector

The fluorescent TSPYL5-EGFP construct used for transient transfection was based on the pTag-GFP2-C vector (Evrogen, Moscow, Russia). The TSPYL5 gene was isolated using a cDNA obtained by reverse transcription of mRNA isolated from the blood of a healthy donor. The isolated genes were amplified using corresponding primers and then cloned into the pTag-GFP2-C vector through restriction–ligation. TSPYL5-EGFP fragments were obtained by PCR using Q5 polymerase (NEB, Ipswich, MA, USA) with the appropriate primers. The pUltraHot-Fucci construct was amplified from the Fucci reporter pCCL-CellCycle plasmid and cloned into the pUltraHot vector using restriction–ligation, in which the mCherry gene was replaced with the puromycin resistance gene. The pCCL-CellCycle plasmid was a gift from John M. Greally (Addgene plasmid #132429), and the pUltraHot plasmid was a gift from Malcolm Moore (Addgene plasmid #24130). Plasmids were prepared using electroporation and Escherichia coli DH5 alpha cells. Several clones were selected for sequencing. Plasmid isolation was performed using the Plasmid Miniprep kit (Evrogen, Russia). All constructs were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the resulting samples were analyzed using the ABI PRISM 3500 genetic analyzer (Applied Biosystems, Foster City, CA, USA).

In Vitro ubiquitination was performed as follows: E1 (2 μg) (Addgene #63571) obtained according to [44 (link)] was mixed with UbcH5c (4 μg) (Addgene #12643) obtained according to [45 (link)], ubiquitin (20 μg), and Ub-TagGFP2 (4 μg). The pET22-based plasmid coding for the Ub-TagGFP2-His₆ was generated by the overlap PCR utilizing the pTagGFP2-C vector (Evrogen, Moscow, Russia) as a matrix. Two terminal glycine residues in the ubiquitin sequence were substituted by valine in order to enhance resistance toward deubiquitination enzymes. The Ub-TagGFP2 was expressed in Escherichia coli (BL21(DE3) strain) and further purified by immobilized metal affinity chromatography (IMAC). The reaction was incubated at 37 °C overnight in a buffer containing 20 mM phosphate buffer (pH 8.0), 100 mM NaCl, 5 mM MgCl₂, 3 mM ATP, and 1 mM DTT. The final volume of the reaction was 65 μL. Furthermore, 5 μL from the in vitro ubiquitination reaction was supplemented with a final concentration of 20 mM phosphate buffer (pH 7.5 or 8.5), 100 mM NaCl, 5 mM MgCl₂, 3 mM ATP, 1 mM DTT, purified 26S proteasome (10 μg), and spermine (10 mM). The reaction volume was adjusted to 20 μL. The reaction was incubated at 37 °C overnight.