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Author
Sánchez Berenguer, Daniel
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Abstract
Autophagy is an evolutionary conserved catabolic process known as an intracellular degradative system that utilizes the autophagosomes to deliver cytoplasmatic components, such as misfolded proteins, damaged and senescent organelles, to the lysosome. Here, we study the function of Protein Interacting with C Kinase 1 (PICK1) in the lysosomal and autophagosomal pathway.
Based on previous findings, we aimed to characterize its function through different experimental assays. First, I studied the expression level of autophagic related proteins, between two different mice groups, global knockout (KO) of PICK1 and wildtype (WT) using western blot.
Furthermore, I used two fluorescent probes termed GFP-LC3-RFP-LC3ΔG, one serving as an internal control, and the second to evaluate the autophagic flux in HEK293 cells. Cells were co-transfected with the GFP-LC3-RFP-LC3ΔG construct together with either a myc-PICK vector, making HEK293 cells PICK1 positive or an empty vector (pDNA3.1+), as control. The probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy as LC3 normally is, while RFP-LC3ΔG remains in the cytosol. The autophagic flux has been calculated through the GFP/RFP signal ratio and further compared with the presence, or absence of PICK1. This study has been done under normal and nutrient depletion conditions, to induce autophagy.
Interestingly, we prove the increasing effect of the autophagic flux when PICK1 is present in HEK293 cells, compared to the control.
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