The NIPA1 (NM_144599.4) tagged with FLAG in C-ter and cloned in pcDNA3.1 vector was acquired from GenEZ™ ORF cDNA Clones (GenScript, Piscataway, NJ, USA). The NIPA1 p.P91R and p.G106R missense mutations were introduced using a Site-Directed Mutagenesis kit (Agilent, Santa Clara, CA, USA) using specific primers for each mutation (available upon request). HeLa cells were grown in complete DMEM and transiently transfected with 2 µg of NIPA1 wild-type (WT), p.P91R, or p.G106R with FuGENE HD Transfection Reagent (Promega, Madison, WI, USA). After 24 h, the cells were fixed, permeabilised and blocked as previously described [13 (link)]. The samples were incubated with the primary antibodies anti-FLAG (Sigma-Aldrich, Saint Louis, MO, USA) and anti-Na+/K+-ATPase, anti-mannose-6-phosphate receptor (M6PR), anti-glucose-regulated protein 94 KDa (GRP94), or anti-early endosome antigen 1 (EEA1; Abcam, Cambridge, UK). The following day, they were exposed to the appropriate secondary antibodies conjugated with fluorophores (Invitrogen, Carlsbad, CA, USA) and examined using the SP2-Leica confocal microscope (Leica, Wetzlar, Germany).
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