Pmaxgfp

1 × 10⁷ BCBL-1 cells were grown in antibiotic-free RPMI. The next day, they were pelleted and rinsed twice in PBS. The cell suspension was transferred into a 0.4 cm electrode gap cuvette with 10 μg of TrueORF Gold pCMV6-Entry DNA vector (Origene, Rockville, MD, USA) containing the Myc-DDK (FLAG)-tagged helicase of interest (Origene, Rockville, MD, USA) or pmaxGFP (AddGene, Cambridge, MA, USA). Cuvettes were treated 2 times at 250 V, 950 μF with the Gene Pulser Xcell device (Bio-Rad, Hercules, CA, USA). Electroporated BCBL-1 cells were grown for 48 h in non-selective RPMI medium. After the 48 h, media was replaced with RPMI containing 400 μg/mL G418 for 4 weeks.

In each assay, MEFs were transiently transfected with localization-free fluorescence-expressing plasmid DNA. The expression plasmids pEGFP-C1 (lysosomal degradable GFP), pMaxGFP (acid-proof GFP) and pTagRFP-C (acid-proof RFP) were purchased from Addgene, Lonza, and Evrogen, respectively. The transfection efficiency was more than 75%, as assessed by the fluorescence of the cells. A few days after transfection, cells with moderate expression (without any aberrant aggregations; see first panel of Fig. 1B) were selected and fluorescence images before photobleaching were obtained using an LSM710 confocal laser-scanning microscope (Zeiss). Almost half of the total cell area was set as the region of interest (ROI) for photobleaching (excitation output level: 100%; iteration: 10–50) using the ZEN application (Zeiss). Subsequently, fluorescence images were obtained after adequate intervals for cytosolic background-equilibration (about 2 min). Note that brighter cytosolic fluorescent-protein expression and sufficient photobleaching enables the acquisition of images with better signal/noise contrast.