Autophagy is one of the key cellular processes responsible for maintaining homeostasis
within the cell. Autophagy is induced in cases of nutrient deficiency, accumulation of
aggregated proteins, malfunctioning organelles, presence of exogenous pathogens, stress,
etc. During autophagy, the captured cargo (proteins, damaged mitochondria and other
organelles …) is degraded in a vesicle called the autolysosome, and the macromolecules
are released into the cytoplasm, where they can be reused in anabolic processes.
Autophagy can, among others, be monitored through two markers, LC3 and p62. LC3 is
a transmembrane protein found in the membrane of the autophagosome and, during
autophagy, changes from the LC3-I form to the LC3-II form. The latter form is lipidated.
p62 is a protein that recruits the cargo intended for degradation via autophagy and anchors
itself to the inner membrane of the autophagosome. To study autophagic activity, inducers
such as rapamycin and starvation are used, as well as degradation inhibitors like
bafilomycin A1 and chloroquine. We attempted to induce autophagy by adding
rapamycin and through starvation using EBSS medium. To inhibit degradation in the final
step of autophagy, we used bafilomycin A1 and various concentrations of chloroquine.
Treatments were applied for different spans of time. We tested two different lysis buffers,
RIPA and 2× SDS with reducing agent, and performed western blot transfer using transfer
buffer with 10% or 20% methanol, onto nitrocellulose and PVDF membrane,
respectively. By comparing fresh and frozen cell lysates, we assessed the effect of
freezing on the levels of selected autophagy markers. It turned out that the best results are
obtained when using rapamycin as an inducer and 100 μM chloroquine as an inhibitor.
The 2× SDS lysis buffer with reducing agent performed better than RIPA, and fresh cell
lysates are more suitable for analysis than frozen ones. Transfer to the membrane is more
efficient with 20% methanol transfer buffer and a PVDF membrane. Fibroblasts express
relatively the highest amounts of both markers, while HEK293T cells are most suitable
for protocol establishment and preliminary experiments.
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