Subcellular Localization of Recombinant RBPR2 in NIH3T3 Cells

Cell lines were grown on coverslips and fixed in a freshly prepared mixture of 4% formaldehyde in 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, and 2 mM potassium phosphate monobasic, pH 7.4) for 30 min at room temperature and processed as previously described (Lobo et al., 2010 (link); Lobo et al., 2013 (link); Solanki et al., 2020 (link); Solanki et al., 2021 (link)). Parental NIH3T3 cells were transiently transfected with the pRbpr2-V5 plasmid, as described previously (Solanki et al., 2020 (link)). Subcellular localization of the recombinant mouse Rbpr2-V5 in NIH3T3 cells was achieved by exposure to the anti-V5 primary antibody (which detects the V5-tagged RBPR2) followed by the anti-rabbit conjugated Alexa 488 secondary antibody staining (Invitrogen, Carlsbad, CA). Cells were examined under a Zeiss LSM 510 UV Meta confocal microscope with an HCX Plan × 40 numerical aperture 1.4 oil immersion objective lens (Zeiss, Jena, Germany). Images were acquired with the Zeiss confocal software, version 2.0. All experiments were carried out in triplicate. Approximately 55–75 cells from 7–9 fields were imaged/counted per experiment (Lobo et al., 2010 (link); Lobo et al., 2013 (link); Rohrer et al., 2021 (link)).