Western Blot Analysis of MAGEA4 Protein

Cellular, vesicular, or purified protein samples were suspended in Laemmli buffer and denatured for 10 min at 100°. The lysates were separated electrophoretically using 10% SDS-PAGE gel and blotted onto a PVDF membrane using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad Laboratories; Hercules, CA, USA). Affinity-purified rabbit polyclonal antibodies against MAGEA4 (2.5 mg/mL) [53 (link)] were used for immunoblotting at dilutions of 1:10,000. Alpha-tubulin (dilution 1:4000; T5168; Merck; St. Louis, MO, USA), anti- TSG101 (dilution 1:10,000, T5701; Merck; St. Louis, MO, USA), anti-E2Tag antibody 5E11 (dilution 1:10,000; Icosagen; Tartu, Estonia) were used in different experiments. Goat anti-rabbit (1 mg/mL, LabAS; Tartu, Estonia) and goat anti-mouse (1 mg/mL, LabAS) antibodies conjugated with HRP were used as secondary antibodies at a dilution of 1:10,000. Protein signals were detected using ECL Western blotting (GE Healthcare; Marlborough, MA USA) reagents. The staining of SDS-PAGE gels was performed with PageBlue Protein Staining Solution (Thermo Scientific; Waltham, MA, USA).