*4.10. Protein Isolation and Western Blot*

Cells for Western Blot analysis were harvested and lysed in a whole-cell lysis buffer composed of 1% Triton-X 100, 10 mM Tris-HCl, pH7.4, 150 mM NaCl and 5 mM EDTA. Prior to use, the lysis buffer was supplemented with phosphatase and protease inhibitor cocktail (phosStop, cOmplete mini, EDTA free, Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was determined by the standard Bradford assay. The normalized samples were immunoblotted as previously described [59] and incubated with primary antibodies for the proteins of interest (Table S3). The secondary antibodies were diluted in a Tris pH 8.0, 0.1% Tween 20 buffer and incubated for 1 h at room temperature. Bound antibodies were detected by the horseradish peroxidase chemiluminescent substrate LuminataTM (Millipore Corporation, Burlington, MA, USA). X-ray films (GE Healthcare, Frankfurt am Main, Germany) were used for chemiluminescence detection. The levels of protein expression on the blot were quantified using ImageJ Software (http://rsbweb.nih. gov/ij) (accessed on 15 March 2021).
