Pnlf1 n cmv hygro

An attR1-ccdB-attR2 cassette (from pBWH) was generated by PCR and cloned into the NotI site of pHTN HaloTag^® CMV-neo (Promega) by Gibson assembly to generate pHTNW. An attR1-ccdB-attR2 cassette (from pAWH) was generated by PCR and cloned into the NotI site of pNLF1-N [CMV/Hygro] (Promega) by gibson, to give pNLF1W. Indicated ORFs were cloned into either pHTNW or pNLF1W by Gateway LR reaction (Thermo Fisher Scientific) following the manufacturer’s indications. HEK293T cells were plated in 24-well plates at a density of 1.4 × 10⁵ cells per well. Cells were transfected with 500 ng of pHTNW-ORF, 5 ng of pNLF1W-ORF, 0.75 μl of Lipofectamine 3000 and 1 μl of P3000 Reagent (Thermo Fisher Scientific). After 20 h, each transformation was re-plated in four wells of 96-well plates at a density of 1 × 10⁴ cells per well for duplicate control and experimental samples (technical replicates), and PPIs were analysed with NanoBRET™ Nano-Glo^® Detection System kit (Promega) following manufacturer’s instructions. Each transformation experiment was performed at least twice. The corrected NanoBRET ratio was calculated according to the manufacturer’s instructions.

Human PALLD, MYPN, FHOD1, and ANKRD1 cDNAs, isolated by PCR using available constructs or a human cDNA library as a template, were cloned into pGBKT7 DNA-BD (Takara Bio), pGADT7-AD (Takara Bio), pFN21A HaloTag CMV Flexi (Promega), pNLF1-N [CMV Hygro] (Promega), and pNLF1-C [CMV Hygro] (Promega) vectors using restriction cloning or the In-Fusion HD Cloning Plus kit (Takara Bio) according to the manufacturer’s instructions. Primer sequences are listed in Supplementary file 1. All constructs were confirmed by sequencing.