The turnover rate of α-actinin assessed in a pulse-chase experiment.

NRVM were transduced with lentiviral vector harboring C-terminal HaloTag labeled α-actinin. 48 hr after transduction, cells were stained with excess TMRDirect (red fluorescence), a cell permeable ligand which forms a stable covalent bond with HaloTag α-actinin. At specified time points after TMRDirect staining as indicated, a different Halo-tag ligand (R110Direct, green fluorescence) was used to stain de novo synthesized HaloTag α-actinin. Both red and green fluorescence were assessed simultaneously. Any newly synthesized α-actinin in different time intervals after TMRDirect staining would be labeled with R110Direct. Representative images were taken under identical exposure parameters. Slight variations in background fluorescence between durations could be due to cell-to-cell variation, differing locations on cover slips, slight variation in depth of mounting media, or to changes in focus.