Abstract
We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes.
Keywords:
Autophagy; Baf. A1, Bafilomycin A1; EBSS, Earle׳s Balanced Salt Solution; FBS, Fetal bovine serum; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; HFs, Human fibroblasts; LC3; LC3, Microtubule-associated protein 1 light chain 3; MEFs, Mouse embryonic fibroblasts; PBS, Phosphate-buffered saline; SB, Sample buffer; SDS, Sodium dodecyl sulfate; TBST, Tris-buffered saline with Tween 20; Western-blot; p62; qPCR, Quantitative PCR.