2.2. Plasmid Construction and Cell Transfection

Plasmids for expression of fluorescently tagged proteins were constructed by standard molecular cloning techniques. Preparation of plasmids for NPMwt and NPMmutA expression was described in [53], for NPMmutE expression was described in [35] and for Δ117wt in [27]. For cloning, DNA fragments corresponding to specific NPM1 and TP53 transcript variants (RefSeq. NM 002520.7, https://www.ncbi.nlm.nih.gov/nuccore/NM_002520.7 and NM 000546.6, https://www.ncbi.nlm.nih.gov/nuccore/NM_000546.6, respectively, from NCBI database accessed on 25 April 2021) were PCR-amplified from cDNA library (Jurkat cells, OriGene, Rockville, MD, USA) or from plasmid pBI-p53 wt/EGFP (Plasmid #16543, Addgene, http://n2t.net/addgene:16543; RRID: Addgene_16543, accessed on 7 June 2021) using extended primers and subsequently they were subcloned to vectors peGFP-C2 or pmRFP1-C2 (originally Clontech, Saint-German-en-Laye, France) using XhoI and BamHI unique restriction sites (Thermo Scientific, Waltham, MA, USA) and T4 DNA ligase (NEB, Ipswich, MA, USA). Plasmids for Δ117mut bearing mutation type A were prepared according to [27,53] by combination of primers used for construction of the single mutants to obtain the desired double mutant. The pBI-p53 wt/EGFP plasmid was a gift from Bert Vogelstein (Addgene plasmid # 16543) [17].

The constructed plasmids were amplified in E. coli competent cells and purified with the PureYield Plasmid Miniprep System (Promega, Madison, WI, USA). HEK-293T cells were seeded to the cell density of 1 × 10⁵/mL 24 h prior transfection and then transfected with jetPrime transfection reagent (Polyplus transfection, Illkirch, France) according to the manufacturer’s protocol. Growth medium was replaced 4 h after the transfection and cells were further grown for 20–40 h prior analysis. NPM and p53 constructs appearing in this study are listed in Table 1.

List of constructs. All protein constructs except PAK2_eGFP are tagged on their N-terminus. Symbol “/” denotes mixture of constructs in text of the article.

¹ See [36] for the description of the NPM mutant type A (NPMmutA). ² See [54] for the description of plasmid for expression of PAK2_eGFP.