HeLa culture, transfection, and labeling of Halo-fusion proteins

High-throughput, predictable systems that are easily modulated are ideal for the study of cell biology. Here we developed a protocol to investigate the role of the Nuclear Factor kappa-B Effector Molecule (NEMO) in Parkin-dependent mitochondrial clearance. Transient transfection of fluorescent constructs allowed us to visualize subcellular structures and dynamics while maintaining flexibility in a consistent model system. The EGFP-NEMO plasmid was repeatedly employed to study NEMO interactions during mitophagy, and we were also able to edit the construct to create both a NEMO point mutation and a Halo-tagged NEMO construct, which we readily expressed in HeLa cells. Halo-fusion constructs, including NEMO and OPTN used in our study, allowed us to visualize the exogenously expressed proteins conjugated to chemical ligands in a variety of colors. This and the accompanying protocols were critical to our characterization of NEMO’s involvement in mitophagy.