Study of Protein-protein Interactions in Autophagy Research

Abstract

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy

“…Cells were tested negative for mycoplasma. Transient transfections of HEK 293 T and HeLa cells were achieved using the calcium-phosphate precipitation method according to standart protocols [61]. SiRNA mediated knockdown experiments performed by using non-targeting siRNA (siCNT) (Dharmacon SiGENOME, D-001210-01-20) or PSMA7 targeting siRNA (siP7) (Dharmacon SiGENOME Smart Pool SiRNA PSMA7, M-004209-00-0010) purchased from Thermo Scientific.…”

“…Immunoprecipitation experiments were performed as previously described [61]. In brief, 2×10 6 cells were harvested 48 or 72 h after transfection and washed with ice-cold 1×PBS.…”

“…Immunofluorescence-based microscopy analyses were performed as previously described [61,63]. Briefly, HeLa and HEK 293 T cells were seeded onto poly-L-lysine coated cover slides.…”