Immunoblotting Procedure for Protein Analysis

The immunoblotting procedure was described in detail in earlier publications [15 (link),16 (link),17 (link),18 (link)]. An amount of 20–40 μg of total cellular protein quantified with the DC Protein-Assay Kit (BioRad) was fractionated by SDS-PAGE on mini-PROTEAN TGX any-kD precast gels (BioRad) and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Eschborn, Germany) equilibrated with methanol and transferred to blotting buffer. After blotting, membranes were blocked with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% bovine serum albumin. Following overnight incubation with the primary antibody at 4 °C in TBST, the primary antibody was removed by washing the blots with TBST. Subsequently, blots were incubated with the appropriate peroxidase-conjugated secondary antibodies and developed with the chemiluminescent detection kit (Amersham ECL Prime Detection Reagent, Cytiva, Marlborough, MA, USA) following the manufacturer’s protocol on a ChemiDoc XRS imaging system (BioRad). Signal quantifications for the proteins of interest and HSP90 were done by densitometry using either the built-in function of the ChemiDoc XRS system or the program Image Lab 5.2.1. The antibodies used are listed in Section 2.1.