Figure 2

Examples of intracellular localized epitope-tagged proteins. (A) Nuclear localized proteins (Heterogeneous ribonucleoprotein A1: HRNPA1, chromosome condensation1: CHC1). Their nuclear localization was confirmed by co-staining with DAPI. In the case of HRNPA1, two clones that incorporated the epitope-tag in the two distinct part of the proteins were isolated. However, both of these epitope-tagged HNRPA1 (N1B1 and N1A10) exhibited the identical localization pattern. (B) Cytoskeletal protein. Beta-actin was epitoped tagged. The co-staining of the cells with anti-myc antibody and FITC-phalloidin confirmed the colocalization. (C) Mitochondrial proteins (F1-beta-ATPase: ATP5B, phosphate carrier precursor isoform 1b: PC). The epitope-tagged ATB5B and endogenous ATB5B proteins exhibited the identical localization as shown by co-staining the cells with anti-myc and anti-ATP5B antibodies. Since no useful antibodies against PC are available, the cells were co-stained with anti-myc and Mitotracker Green FM (Molecular Probes). Both anti-myc and Mitotracker FM showed identical staining, confirming the mitochondrial localization of epitope-tagged PC protein. All of the colocalization studies were preformed with confocal microscopy. Only the cells that underwent the successful Cre-mediated excision express the myc-tagged proteins and were stained with 9E10 antibody. The efficiency of Cre-mediated excision varied greatly depending on the target gene (<0.5% – 2%).