Eosinophil priming and immunoblotting

Eosinophils were primed with cytokines for 20 h and were seeded on HA-IgG for the indicated times. Treatment with bafilomycin-A1 (BioViotaca, Liestal, Switzerland) started after priming and 20 min before seeding on IgG. Cells were lysed directly in LaemmLi buffer (10% SDS plus ß-mercaptoethanol), before boiling and loading onto 15 to 10% SDS-polyacrylamide gels. Proteins were transferred into a PVDF membrane. Immunoblotting was performed as previously described [22 (link)]. Blots were subsequently incubated with desired primary antibodies: anti-ß-actin from Sigma-Aldrich, anti-phospho-cofilin, anti-SQSTM1 and anti-LC3B from Cell Signaling Technology (Danvers, MA, USA) and anti-phospho-p38 from Genetel Laboratories (Pasadena, TX, USA) (all diluted by 2000-fold in 1× TBS, 0.1% Tween-20 with 5% BSA). Then, the appropriate HRP-conjugated secondary antibodies (Calbiochem, San Diego, CA, USA) were utilized. Immunoreactive bands were visualized using ECL reagents and GE LAS4000 chemiluminescence imager (GE Healthcare, Chicago, IL, USA) and iBright CL1000 (InVitrogen, ThermoFisher Scientifics, Waltham, MA, USA). Bands were quantified using ImageJ (https://imagej.nih.gov/ij/ accessed on 23 April 2018).