Analysis of myosin light chain migration pattern in 2D gels of human left ventricular proteins - proximity labeling

NIMA-related kinase 9 (NEK9) was identified to interact with the essential myosin light chain (ELC). To validate these findings ascorbate peroxidase (APEX) catalyzed proximity labeling (Hung et al. Nat Protoc. 2016) was performed. To do so, human ELC fused to APEX was overexpressed in HEK293 cells. Empty pcDNA3-APEX2-NES vector serves as negative control. Half of each sample was treated with H2O2, which catalyzes protein labeling in close proximity of ELC. After streptavidin pull down of labeled proteins, the samples were analyzed by mass spectrometry.