2.10. Western Blot for Autophagy Analysis

Western blot was used to investigate the expression and possible modifications of LC3-II involved in autophagy. Immunodetection of LC3-II (0231-100/LC3-5F10, mouse monoclonal; NanoTools, Teningen, Germany) was performed in basal conditions and after 24 h of incubation with poly(I:C) in G0/G0 and G2/G2 podocytes. However, detection of LC3-II only shows a snapshot of protein levels in the cell without reproducing the dynamic information of the autophagic process, so-called autophagic flux. As autophagy is a continuously ongoing process, an increased level of LC3-II may result from either increased production or a decreased degradation of autophagosomes. To assess autophagic flux, cells were treated with 100 nM Bafilomycin A1 (Baf A1) (LC Laboratories, Woburn, MA, USA) or dimethylsulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) as vehicle for 3 h before collecting pellet, as previously described [32]. Baf A1 is a lysosomal inhibitor and autophagic flux blocker. It blocks the fusion of autophagosomes and lysosomes leading to an accumulation of the autophagosomal marker LC3-II. As such, in the presence of Baf A1, information is gathered only about LC3-II (and autophagosome) production. For the blotting, cells were lysed with 50–100 µL of RIPA buffer. The protein concentration was determined using BCA protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). Equivalent amounts of protein were separated on a NuPAGE^TM 4–12% Bis-Tris Protein Gel (Invitrogen, Waltham, MA, USA) and followed by blotting on polyvinylidene difluoride (PVDF) membrane. Primary and secondary antibodies were used according to the manufacturer’s instructions. Blots were developed using HRP-conjugated secondary antibodies and analysed using Image J software. Three independent experiments were performed. Full-length blots are presented in Supplementary Figure S1.