“…Mutant plasmids were obtained from 10 ng of wild‐type pCMV6‐AC‐GFP encoding the full length TRPS1 transcript (RG215856: TRPS1 NM_014112 Human Tagged ORF Clone; OriGene, Rockville, MD, USA) by site‐directed mutagenesis using the QuickChange II XL Site‐Directed Mutagenesis kit (Agilent Technologies, Cedar Creek, TX, USA) and 125 ng of mutation‐specific primers to introduce our novel c.343C>T and the c.422C>T variants, the c.2894G>A variant in exon 7 (mutational hot spot located in the NLS2 domain, used as mutated positive control) and the already described variants c.47G>A and c.203A>C (known variants were renamed according to the reference sequence NM_014112; primer sequences available upon request). ( 25 , 26 , 31 ) Transformed ampicillin‐resistant E. coli colonies were recovered from agar plates and expanded over night and plasmids were purified by the Plasmid Plus Maxi Kit (Qiagen, Valencia, CA, USA) and tested by Sanger sequencing. Twenty thousand (20,000) cells/cm 2 of HepG2 and 15,000 cells/cm 2 of L88/5 were grown (each condition was performed in triplicate) until 80% confluence, transfected with 1 μg of wild‐type and mutated pCMV6‐AC‐GFP TRPS1 expression vectors by the Mega Tran 2.0 transfection reagent (OriGene, Rockville, MD, USA).…”