Transit lt1 reagent

Plasmids containing full-length PBF cDNA with or without a C-terminal haemagglutinin (HA) tag have previously been described (Stratford et al. 2005 (link)). All 10 PBF mutations were recapitulated in both HA-tagged and untagged PBF using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). FLAG-tagged PBF was generated through the ligation of an oligo encoding the FLAG sequence (DYKDDDK) into the pcDNA3.1+_PBF plasmid. The FLAG tag lies within the protein after residue E34. Previous attempts to tag PBF at the N-terminal end were unsuccessful due to the presence of a cleavable signal peptide; to overcome this, the FLAG epitope lies downstream of the cleavage site. The C51R and R140W mutations were also introduced into FLAG-tagged PBF. The NIS cDNA was housed in the pcDNA3.1+ vector with a C-terminal MYC tag (Smith et al. 2009 (link)). For stable transfections, untagged wild-type (WT) and PBF mutant cDNAs were cloned into the pCI-neo vector (Promega). Transfections were performed with TransIT-LT1 reagent (Geneflow, Lichfield, UK) following the manufacturer’s protocol at a 3:1 reagent to DNA ratio and the experiments were performed after 24–48 h.

The full-length human NIS cDNA was cloned in the pcDNA3.1+ vector with a C-terminal MYC (NIS-MYC) or HA (NIS-HA) tag (30 (link)). NIS-MYC and NIS-HA were both required for the coimmunoprecipitation (co-IP) and proximity ligation assays (PLAs), which necessitated two distinct tags. For use in the Förster resonance energy transfer (FRET) experiments, NIS cDNA was inserted into the HindIII and BamHI restriction sites of the cerulean-N1 and citrine-N1 vectors (gifts from Michael Davidson & Dave Piston obtained from Addgene, Watertown, MA) to create NIS constructs conjugated at the C-terminus to a cerulean or citrine fluorophore, respectively.
Transfections were performed with TransIT^®-LT1 reagent (Geneflow, Lichfield, United Kingdom) following the manufacturer's protocol at a 3:1 reagent to DNA ratio and experiments performed after 48 hours. Specific mutations were made as indicated using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA).

Normal rat kidney (NRK) cells and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA, cat D6046) with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin and streptomycin at 37 °C in humidified air with 5% CO₂. For most imaging experiments, cells were plated onto glass coverslips coated with 1% (w/v) poly-l-lysine. For imaging individual lysosomes, HeLa cells were grown on 35 mm glass-bottom dishes (#P35G-1.0-14-C, MatTek Corporation, Ashland, MA, USA) coated with human fibronectin (10 µg/mL, Merck Millipore, Watford, UK). Transfections were carried out 24 h after plating using either PEI (Polysciences, Inc, Warrington, PA, USA) (1 µg DNA/2 µL of 1 mg/mL PEI) or TransIT-LT1 reagent (1 µg DNA/2.5 µL reagent, GeneFlow, Lichfield, UK). Cells were then incubated for 48 h at 37 °C prior to use.